DE69733834T2 - THYROXINE ANALOGS WITH NO SIGNIFICANT HORMONAL ACTIVITY FOR THE TREATMENT OF MALICIOUS TUMORS - Google Patents

Abstract

The present invention provides methods for treating cancer, particularly malignant tumors, with thyroxine analogues having no significant hormonal activity. A thyroxine analogue is administered to an afflicted mammal in an amount effective to cause depression or regression of malignant tumor growth or to treat cancer. Particularly preferred thyroxine analogues are those capable of causing about 35 percent or more inhibition of initial velocity of microtubule protein assembly in vitro.

Description

CROSS REFERENCE RELATED APPLICATIONS

These Registration is a continuation application the likewise pending US application serial no. 08 / 655,267, filed June 4, 1996, the disclosure of which is incorporated herein by reference.

AREA OF INVENTION

The The present invention relates generally to the field of cancer therapeutics. More particularly, the present invention relates to specific thyroxine analogues, which have no significant hormonal activity, in particular Methyl-3,5-diiodo-4- (4'-methoxyphenoxy) benzoate, as strong, selective and non-toxic antitumor agents.

BACKGROUND THE INVENTION

Malignant cancerous Growths are serious because of their unique properties Challenges for modern medicine. These features include uncontrollable Cell proliferation malignant to an unregulated proliferation Fabric leads, the ability, to invade local and even distant tissues, the absence of differentiation, the absence of detectable symptoms and most significant the lack of effective therapy and prevention.

cancer can be in any tissue of any organ in develop at any age, the etiology of cancer is not clearly defined, but mechanisms such as genetic susceptibility, chromosome disruption, Viruses, environmental factors and immunological disorders are all with one malignant cell proliferation and cell transformation Service.

The Antineoplastic chemotherapy currently includes various groups of Drugs, the alkylating agents, purine antagonists and anti-tumor antibiotics lock in. Alkylating agents alkylate cell proteins and nucleic acids, which prevents cell replication, breaks cell metabolism and finally leads to cell death. Typical alkylating agents are nitrogen mustard, cyclophosphamide and chlorambucil. Those associated with an alkylating agent treatment toxicities close nausea, Vomiting, hair loss, hemorrhagic Cystitis, pulmonary fibrosis and an increased risk, an acute one leukemia to develop.

purine, Pyrimidine and folate antagonists are cell cycle and phase specific and, to promote an anti-tumor effect, they require that the cells in the cell replication cycle and in the DNA synthesis phase of replication. The purine antagonists, such as 6-mercaptopurine or 6-thioguanidine, inhibit de novo-purine synthesis and interconversion of purines. The pyrimidine antagonists, such as cytarabine, 5-fluorouracil or floxuridine, inhibit DNA synthesis by inhibiting deoxycytidylate kinase and DNA polymerase.

Folate antagonists, e.g. Methotrexate binds tightly to the intracellular enzyme Dihydrofolate reductase, which ultimately leads to cell death, which from an inability results in synthesizing pyrimidines. The with the use toxicities associated with these compounds include other hair loss, Myelosuppression, vomiting, nausea and cerebellar Ataxia.

Plant alkaloids, such as vincristine and vinblastine, or the podophyllotoxins etoposide and teniposide generally inhibit mitosis and DNA synthesis and RNA-dependent Protein synthesis. The toxicities of this Drugs are similar to those described above and shut down Myopathy, myelosuppression, peripheral neuropathy, vomiting, nausea and hair loss.

Antitumor antibiotics, like doxorubicin, daunorubicin and actinomycin act as intercalators DNA, which prevents cell replication, the synthesis of DNA-dependent RNA inhibits and inhibits the DNA polymerase. Bleomycin causes cleavage The DNA and mitomycin acts as an inhibitor of DNA synthesis by bifunctional Alkylation. The toxicities of these antibiotics are numerous and serious and include necrosis, Myelosuppression, anaphylactic reactions, loss of appetite, dose-dependent cardiotoxicity and pulmonary fibrosis.

Other compounds used for the chemotherapeutic treatment of cancer are inorganic ions, such as cisplatin, modifiers of the biological reaction, such as interferon, enzymes and hormones. All of these compounds are accompanied by toxic side effects similar to those mentioned above.

US 4,724,234 is intended to describe a method for producing oncolysis and regression of malignant tumors and other malignant conditions without side effects on normal body cells. Namely, there is described a caloric and compositional nutritional regimen to be administered concomitantly with drug treatment of an agent or agents which decouple oxidative phosphorylation, most preferably 2,4-dinitrophenol. Thyroxine is also listed as one of the decoupling agents.

consequently it would be extremely beneficial be safe and non-toxic chemotherapeutic compositions to provide tumor cell proliferation and / or neoplastic Effectively inhibit and / or suppress growth. In addition, it would be extremely beneficial to have secure, to provide effective and non-toxic chemotherapeutic compositions, which are easy to administer.

The Identification of safer, more effective, non-toxic and orally administrable organic compounds that reduce malignant tumor growth in mammals or regress can, and the use of such compounds to treat cancer is therefore desirable and the object of this invention.

SUMMARY THE INVENTION

The The present invention relates to specific thyroxine analogs which do not have any have significant hormonal activity, in particular methyl 3,5-diiodo-4- (4'-methoxyphenoxy) benzoate ("DIME") to malignant tumor growth to reduce or regress and to treat cancer. The thyroxine analogs are typical characterized in that they have a significant hormonal Effectiveness is missing.

Especially The present invention relates to a medicament for the treatment a malignant tumor in a mammal containing an amount of the thyroxine analogue sufficient to reduce the growth of the malignant tumor, wherein the thyroxine analogue is characterized by having a Compound is that has an about 35% or higher inhibition of the initial rate microtubule-protein assembly in vitro, preferably about 45% or higher, stronger preferably about 70% or higher and the strongest preferably about 90% or higher Inhibition of the initial rate of microtubule-protein assembly in vitro.

und pharmazeutisch verträglicher Salze davon, wobei:
X = O, S, CH₂, Carboxy oder abwesend;
Y = O oder S;
R₁ = Methyl oder Ethyl;
R₂, R₃, R₄ und R₅ jeweils unabhängig aus H, (C₁-C₄)-Alkyl, (C₁-C₄)-Alkenyl, (C₁-C₄)-Alkinyl, Hydroxy, (C₁-C₄)-Alkoxy und Halogen ausgewählt sind; und
R₆, R₇, R₈ und R₉ jeweils unabhängig aus H, (C₁-C₄)-Alkyl, (C₁-C₄)-Alkenyl, (C₁-C₄)-Alkinyl, Hydroxy, (C₁-C₄)-Alkoxy, Halogen, NO₂ und NH₂ ausgewählt sind.Thyroxine analogs useful in the processes of the present invention are compounds having the structural formula:

and pharmaceutically acceptable salts thereof, wherein:
X = O, S, CH ₂ , carboxy or absent;
Y = O or S;
R ₁ = methyl or ethyl;
R ₂ , R ₃ , R ₄ and R _{5 are} each independently selected from H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxy, (C ₁ -C ₄ ) alkoxy and halogen are selected; and
R ₆ , R ₇ , R ₈ and R _{9 are} each independently selected from H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxy, (C ₁ -C ₄ ) alkoxy, halogen, NO ₂ and NH _{2 are} selected.

und pharmazeutisch verträglicher Salze davon, wobei:
X = O, S, CH₂, Carboxy oder abwesend;
Y = O oder S;
R₁ = Methyl oder Ethyl;
R₂, R₃, R₄ und R₅ jeweils unabhängig aus H, (C₁-C₄)-Alkyl, (C₁-C₄)-Alkenyl, (C₁-C₄)-Alkinyl, Hydroxy, (C₁-C₄)-Alkoxy und Halogen ausgewählt sind; und
R₇ und R₈ jeweils unabhängig aus H, (C₁-C₄)-Alkyl, (C₁-C₄)-Alkenyl, (C₁-C₄)-Alkinyl, Hydroxy, (C₁-C₄)-Alkoxy, Halogen, NO₂ und NH₂ ausgewählt sind.In another illustrative embodiment, thyroxine analogs useful in the methods of the present invention are compounds having the structural formula:

and pharmaceutically acceptable salts thereof, wherein:
X = O, S, CH ₂ , carboxy or absent;
Y = O or S;
R ₁ = methyl or ethyl;
R ₂ , R ₃ , R ₄ and R _{5 are} each independently selected from H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxy, (C ₁ -C ₄ ) alkoxy and halogen are selected; and
R ₇ and R _{8 are} each independently selected from H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxy, (C ₁ -C ₄ ) - Alkoxy, halogen, NO ₂ and NH _{2 are} selected.

In a preferred embodiment In the invention, the thyroxine analogue is methyl 3,5-diiodo-4- (4'-methoxyphenoxy) benzoate ("DIME").

SHORT DESCRIPTION THE FIGURES

1 illustrates the effects on cell morphology after 18-24 hours incubation of E-ras 20 cells with 4 μM DIME. Field A: not drug-treated (control); Field B: drug-treated; Panel C: drug-treated. Fields A and B are enlarged 150 times; Field C is enlarged 300 times.

2 Figure 3 is a graph illustrating the decrease in tumorigenic effect of DIME-treated E-ras transformed bovine endothelial cells; and

3 Figure 9 is a graph illustrating serum half-life ( _t½ ) and oral bioavailability of DIME in mice.

4 shows the tumorigenic effect of a DIME pretreatment (10 μM for 4 days) on tumor formation of 10 ⁵ or 10 ⁶ E-ras 20 cells per inoculum.

5 shows the effect of DIME concentration on the rates of colony formation by MDA-MB-231 cells.

6 shows the induction of DNA breaks by 0.0 μM (a), 2.0 μM (b), 5 μM (c) and 10 μM (d). E-ras cells (2 x 10 ⁴ cells / cm ² ) were treated with DIME 18 hours before the TUNEL assay.

7 shows the thin layer chromatographic separation of DIME (D) and its carboxylic acid degradation product (A) by A-549 (lung cancer) cell extracts (equivalent to 2 x 10 ⁶ cells). In the experiment shown in lanes 1 and 2, the esterase activity of the extract occurs during 4 hours incubation with 1 μM DIME. Lane 2 illustrates esterase inhibition by 125 μM BNPP. Lanes 3 and 4 represent the same experiments as in lanes 1 and 2, except that the cell extracts were prepared from intact cells preincubated with 1 μM DIME for 24 hours, then washed and extracted. This experiment illustrates that the esterase is not induced by DIME.

8th shows the increase in the inhibitory effect of DIME on the growth of A-59 cells by BNPP.

9 graphically depicts the effect of DIME on MDA-MB 231 cells. The initial seeding density was 0.05 x 10 ⁶ per 2 cm ² .

11 provides the flow cytometric analysis performed on human MDA-MB 231 breast cancer cell nuclei, showing that cores having a G2 content of DNA accumulated through 18 hours of drug exposure, indicating M-phase blockage.

12 shows images illustrating a 13-hour delay in mitosis, followed by an irregular division into six daughter cells. Frame 1: 0 hrs: 0 min .; Frame 2: 10:55; Frame 3: 24:20; Frame 4: 24:40; Frame 5: 24:55; Frame 6: 25:10; Frame 7: 26:35; Frame 8: 28:20.

13 shows the effect of DIME on the M-phase residence time of MDA-MB-231 cells.

14 shows the quantitative analysis of the abnormal cell division induced by DIME.

15 shows the effect of DIME on the rate of entry into the M phase of MDA-MB-231 cells.

16 (A, B and C) show the hybridization of chromosomes 19 DNA in DIME-treated MDA-MB-231 cells (metaphase spread). 16A is the control, 16B is the DNA-stained image, 16C is the hybridization of chromosome 19 of the 5-day treatment with 1 μM DIME (metaphase).

17 (A, B and C) show the effect of DIME treatment on the mitotic spindle of MDA-MB-231 cells. Plate A: control (no drug); Plate B: 1 μM DIME for 18 hr .; Plate C: 1 μM DIME for 5 days.

18 shows an optical test for microtubule assembly (MTP polymerization). The concentration of GTP was 1 μM (right curve) and the effect of 4 μM DIME is illustrated in the left curve. Other conditions were the same as described in the legend of Table 13.

19 shows the effect of the increasing concentration of DIME on the initial linear rate of MTP polymerization. The concentration of GTP was 1 mM, other conditions being identical to those given in the legend of Table 13.

20 Figure 3 shows the Michaelis-Menten analysis of the effect of 1 μM DIME (closed circles) on the initial linear rate of MTP polymerization as a function of GTP concentration (open circles, no drug). Other conditions were the same as described in the legend in Table 13.

DETAILED DESCRIPTION OF THE INVENTION

definitions

As used here:
"Alkyl" refers to a saturated branched, straight-chain or cyclic hydrocarbon radical. Typical alkyl radicals include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, cyclobutyl, tert-butyl, pentyl and hexyl.
"Alkenyl" refers to an unsaturated branched, straight-chain or cyclic hydrocarbon radical having at least one carbon-carbon double bond. The rest can be present either in the cis or trans conformation on the double bond (s). Typical alkenyl radicals include ethenyl, propenyl, isopropenyl, cyclopropenyl, butenyl, isobutenyl, cyclobutenyl, tert-butenyl, pentenyl and hexenyl.
"alkynyl" refers to an unsaturated branched, straight-chain or cyclic hydrocarbon radical having at least one carbon-carbon triple bond. Typical alkynyl radicals include ethynyl, propynyl, butynyl, isobutinyl, pentynyl and hexynyl.
"Alkoxy" refers to a radical -OR, wherein R is alkyl, alkenyl or alkynyl as defined above.
refers to "halogen" fluoro, chloro, bromo and iodo substituents.
"mammal" refers to animals or humans.
"pharmaceutically acceptable salt" refers to those salts of compounds which retain the biological activity and properties of the free bases and which are obtained by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid become. Pharmaceutically acceptable salts include, for example, alkali metal salts such as sodium and potassium, alkaline earth metal salts and ammonium salts.
"Pharmacophore" refers to the critical three-dimensional arrangement of molecular entities or fragments (or the electron density distribution) recognized by a receptor (Burger's Medicinal Chemistry and Drug Discovery Bd.I: Principles and Practice 619, 5th Ed., John Wiley & Sons, New York).
"therapeutically effective amount" refers to an amount of a compound or composition that is effective to reduce, suppress, or regress malignant cell growth, or to ameliorate the symptoms associated with cancer.

Description, more specific embodiments

The The present invention relates to medicaments for treating malignant Tumors and cancer in mammals with analogs of thyroxine, which are characterized in that they have no significant hormonal activity. Preferably the present invention is based in part on the surprising finding that certain analogues of thyroxine, which have no hormonal activity show strong, selective and non-toxic inhibitors of malignant tumor growth are. The preferred thyroxine analog is referred to herein as DIME.

thyroxine, an amino acid the thyroid gland (Merck Index, 1989, 9348: 1483), and thyroxine analogs are in the art well known. It is well established in the literature that thyroid hormones, specifically the thyroxins T3 and T4, two different types of biological Have effects: one on the cell metabolism, the second on Cell Differentiation and Development (Jorgensen, 1978, "Thyroid Hormones and Analogues II. Structure-Activity Relationships, "In: Hormonal Proteins and Peptides, Vol. VI, pp. 107-204, C.H. Li, ed., Academic Press, NY). For example, suppresses thyroxine the uptake of iodine by the thyroid (Money et al., 1959, "The Effect of Various Thyroxine Analogues on Suppression of Iodine-131 Uptake by the Council Thyroid, "Endocrinology 64: 123-125) and induces cell differentiation as studied by tadpole metamorphism (Money et al., 1958, "The Effect of Change in Chemical Structure of Some Thyroxine Analogues on the Metamorphosis of Rana Pipiens Tadpoles, "Endocrinology 63: 20-28.) Also suppress thyroxine and certain thyroxine analogues do not make the growth more malignant thyreotropic Murine pituitary tumors (Kumaoka et al., 1960, "The Effect of Thyroxine Analogues on a Transplantable Mouse Pituitary Tumor, "Endocrinology 66:" 32-38; Grinberg et al., 1962, "Studies with Mouse Pituitary Thyrotropic Tumors. V. Effect of Various Thyroxines Analogs on Growth and Secretion, "Cancer Research 22: 835-841). The structural requirements of thyroxine and thyroxine analogues for one metabolic stimulation and induction of cell differentiation not identical (see Jorgensen, 1978, "Thyroid Hormones and Analogues II. Structure Activity Relationships, "In: Hormonal Proteins and Peptides, Vol. VI, p. 150, C.H. Li, eds., Academic Press, NY). For example, Money et al. found that there is no correlation between suppression of thyroid iodine uptake and induction of tadpole metamorphism (Money et al., 1958, "The Effect of Change in Chemical Structure of Some Thyroxine Analogues on the Metamorphosis of Rana Pipiens Tadpoles, "Endocrinology 63: 20-28).

Based on these observations was designed that previously unknown Cell reactions by certain thyroxine analogues, neither Modes of action (metabolically or differentially) of thyroxine T3 and T4 are shown, shown, altered or induced can.

The connections

und pharmazeutisch verträgliche Salze davon, wobei:
X = O, S, CH₂, Carboxy oder abwesend;
Y = 0 oder S;
R₁ = Methyl oder Ethyl;
R₂, R₃, R₄ und R₅ jeweils unabhängig aus H, (C₁-C₄)-Alkyl, (C₁-C₄)-Alkenyl, (C₁-C₄)-Alkinyl, Hydroxyl, (C₁-C₄)-Alkoxy und Halogen ausgewählt sind; und
R₆, R₇, R₈ und R₉ jeweils unabhängig aus H, (C₁-C₄)-Alkyl, (C₁-C₄)-Alkenyl, (C₁-C₄)-Alkinyl, Hydroxyl, (C₁-C₄)-Alkoxy, Halogen, NO₂ und NH₂ ausgewählt sind.Thyroxine analogs useful in the present invention are compounds having the structural formula:

and pharmaceutically acceptable salts thereof, wherein:
X = O, S, CH ₂ , carboxy or absent;
Y = 0 or S;
R ₁ = methyl or ethyl;
R ₂ , R ₃ , R ₄ and R _{5 are} each independently selected from H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxyl, (C ₁ -C ₄ ) alkoxy and halogen are selected; and
R ₆ , R ₇ , R ₈ and R _{9 are} each independently selected from H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxyl, (C ₁ -C ₄ ) alkoxy, halogen, NO ₂ and NH _{2 are} selected.

und pharmazeutisch verträgliche Salze davon, wobei:
X = O, S, CH₂, Carboxy oder abwesend;
Y = O oder S;
R₁ = Methyl oder Ethyl;
R₂, R₃, R₄ und R₅ jeweils unabhängig aus H, (C₁-C₄)-Alkyl, (C₁-C₄)-Alkenyl, (C₁-C₄)-Alkinyl, Hydroxyl, (C₁-C₄)-Alkoxy und Halogen ausgewählt sind; und
R₇ und R₈ jeweils unabhängig aus H, (C₁-C₄)-Alkyl, (C₁-C₄)-Alkenyl, (C₁-C₄)-Alkinyl, Hydroxyl, (C₁-C₄)-Alkoxy, Halogen, NO₂ und NH₂ ausgewählt sind.In a preferred embodiment, thyroxine analogs useful in the methods of the present invention are compounds having the structural formula:

and pharmaceutically acceptable salts thereof, wherein:
X = O, S, CH ₂ , carboxy or absent;
Y = O or S;
R ₁ = methyl or ethyl;
R ₂ , R ₃ , R ₄ and R _{5 are} each independently selected from H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxyl, (C ₁ -C ₄ ) alkoxy and halogen are selected; and
R ₇ and R _{8 are} each independently selected from H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxyl, (C ₁ -C ₄ ) - Alkoxy, halogen, NO ₂ and NH _{2 are} selected.

In a particularly preferred embodiment the thyroxine analog is methyl 3,5-diiodo-4- (4'-methoxyphenoxy) benzoate ("DIME").

thyroxine analogues like DIME have been described in the literature. However, that became reported that DIME unlike thyroxine no significant metabolic or cell-differentiating efficacy (as determined by Tadpole metamorphosis) (Money et al., 1958, "The Effect of Change in Chemical Structure of Some Thyroxine Analogues on the Metamorphosis of Rana Pipiens Tadpoles, "Endocrinology 63: 20-28; Stasilli et al., 1959, "Antigoitrogenic and Calorigenic Activities of Thyroxine Analogues in Rats, "Endocrinology 64: 62-82). For example, the uptake of iodine into the thyroid gland of rats only slightly (15%) inhibited by DIME compared to thyroxine (Money et al., 1959, "The Effect of Various Thyroxine Analogues on Suppression of Iodine-131 Uptake by the Rat Thyroid, "Endocrinology 64: 123-125). Furthermore It has been reported that DIME has no inhibitory activity against the Growth of a non-malignant mouse pituitary adenoma (Kumaoka et al., 1960, "The Effect of Thyroxine Analogues on a Transplantable Mouse Pituitary Tumor, "Endocrinology 66: 32-38; Grinberg et al., 1962, "Studies with Mouse Pituitary Thyrotropic Tumors. V. Effect of Various Thyroxines Analogs on Growth and Secretion, "Cancer Research 22: 835-841). No studies with malignant cells have been reported.

It it has now been found that certain thyroxine analogs, which have no significant hormonal activity, in particular DIME, not just the growth of a variety of malignant cell types (see Table 3), but also a tumor cell apoptosis, a Microkernel formation has been induced. This cytostatic and cytocidal effects are structurally sensitive. The examination of thirteen structural analogues and homologues of DIME shows that even minor changes the methyl ester and 4'-methoxy substituents the molecule completely make ineffective. While DIME in both cell assays As well as being highly effective in vivo, the 4'-propoxy and ethyl ester homologs are completely ineffective. Therefore, DIME defines a crucial array of molecular Units or a pharmacophore with special cytostatic and cytocidal effect and consequently a significant chemotherapeutic Potential.

Even though is not intended to be bound by theory, believes one, that the most probable molecular mode of action here Thyroxin analogues described cell cycle inhibition and induction of Apoptosis is.

The Progression of eukaryotic cells through the cell division cycle mainly by the effectiveness cyclin-dependent Controlled protein kinases. The best studied event is the transition from G2 to M-phase produced by cdc2 kinase complexed with cyclin B, is controlled (for an overview see Dunphy, 1994, Trends Cell. Biol. 4: 202-207). cdc2 kinase activation requires phosphorylation, a process regulated by protein phosphatase 2A (for a review see Wera & Hennings, 1995, Biochem. J. 311: 17-29).

It has been found that the thyroxine analogs described herein exert specific activation of protein phosphatase 2A both in vitro and in vivo. In vivo, activation of protein phosphatase 2A coincides with inhibition of cdc2 kinase and dephosphorylation of MAP kinase and topoisomerase II, rendering the latter two enzymes ineffective. DIME has no metabolic activity, it also does not inhibit the biosynthetic pathways of DNA, RNA or proteins. Thus, the most likely mode of action cell cycle inhibition and induction of apoptosis via dephosphorylation of these crucial regulatory proteins. Thus, activation of phosphatase 2A and concomitant cdc2 kinase inhibition is an important and powerful therapeutic target for the treatment of cancer.

Although changes at the ester and 4'-positions The efficacy of DIME seems to be significantly affected Thyroxine analogues that are used to reduce malignant tumor growth and Treating cancer are useful, not limited to DIME. To the Example shows the 4'-ethoxy homologue about 25-30% maximum cytocidal effect on human cancer cells compared to DIME (Example 4). It is also expected that DIME on the aromatic Ring positions or at the bridge oxygen can be substituted without significant loss of efficacy.

It it is known that the aromatic rings of thyroxine are not within same level (Jorgensen, 1978, "Thyroid Hormones and Analogues II. Structure-Activity Relationships, "In: Hormonal Proteins and Peptides, Vol. VI, pp. 107-204, C.H. Li, ed., Academic Press, NY). It is also known that the Ring positions of both aromatic rings in thyroxine with a variety to substituents which include alkyl, halogen, nitro and amino radicals, with different dimensions substituted on maintaining hormonal efficacy (ibid.) could be. Furthermore may be missing the connecting the rings ether oxygen or with a Variety of residues or atoms that the aromatic rings do not restrict to the same level such as a methylene group, a carboxy group or sulfur, be replaced without a significant loss of hormonal activity (Ibid.). Consequently, it is expected and predictable that similar Substitutions to DIME no significant loss of efficacy against cancer. Significantly, the 2'-chloro analogue of DIME about 25% maximum inhibitory effect on the growth of human Cancer cells compared to DIME (Example 5).

by virtue of the strict correlation between in vitro and in vivo efficacy (see Examples 2-7) can effective compounds useful in the methods of the invention are, suitably be identified in in vitro assay screening assays. Such tests can on the ability a particular compound to activate protein phosphatase 2A, check, as in examples 2-3 is described. Typically, compounds that are used in the Methods of the present invention are useful, the protein phosphatase Increase 2A efficiency by a factor of about two to three, as measured by the assay described in Example 2 or 3 becomes.

Such assays may also test for the ability of a particular compound to inhibit malignant tumor cell growth in vitro or in vivo or to abrogate the tumorigenic effect of malignant cells, as described in Examples 4-6. In general, active compounds useful in the methods of the present invention will have an I ₅₀ value (concentration of the compound that is lethal to 50% of a cell culture compared to a control culture) in the range of about 0.5 μm to 5 μm 0 μm, as measured by the assay described in Example 4.

As it goes without saying to a specialist can be used many types of malignant tumor cell cultures and cell lines to be effective, the HL-60, HT-144, E-ras-20, DU-145, MDA-168, MCF-7, 855-2 and MDA-MB-231 lock in, but not limited to that are. Naturally can also, as the expert of course other in vitro and / or in vivo assays for efficacy to be used against tumors and / or to test for cancer to identify effective thyroxine analogues present in the present Invention are usable.

The chemical formulas mentioned here may be the phenomena of tautomerism or Show conformational isomerism. Since the formula drawings within This description only one of the possible tautomeric or conformational isomers Can represent forms, should of course be that the invention any tautomeric or conformational isomers Forms includes, the similar show biological or pharmacological activities such as DIME, as they are described here.

In addition to the compounds described above and their pharmaceutically compatible salts If appropriate, the invention may be solvated or unsolvated Use forms of the compounds (e.g., hydrated forms).

The compounds described herein can be prepared by any method known to be applicable to the preparation of chemical compounds. Suitable methods are illustrated by the representative examples. Necessary starting materials can by standard Verfah ren organic chemistry.

cancerous tumors

The thyroxine analogues described herein are for the treatment of a broad Variety of cancerous ulcers usable. Such cancerous ulcers shut down as an example and not as a limitation of carcinomas such as pharynx, Colon, rectal, pancreatic, stomach, liver, lung, Breast, skin, prostate, ovarian, cervix, uterus and bladder cancer ulcers, leukemias, Lymphomas, gliomas, retinoblastomas and sarcomas.

In a preferred embodiment In the invention, cancer is associated with the formation of solid tumors, as an example and not as a restriction mammary and Prostate ulcers lock in.

Pharmaceutical formulations and administration routes

One Thyroxine analogue useful in the present invention may include Human patients in the form of a pharmaceutically acceptable Salt or in the form of a drug, wherein the connection with suitable carriers or excipients in a therapeutically effective amount, i. in Doses effective to reduce malignant cell growth or suppress or to improve the symptoms associated with cancer respectively, is mixed.

routes of administration

The thyroxine analogues and drugs described herein can be used by a variety of ways are administered. Suitable routes of administration can for example, oral, rectal, transmucosal or intestinal administration, parenteral delivery, including intramuskukärer, subcutaneous, intramedullary Injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, include intranasal or intraocular injections.

In another embodiment The connection can be more local than systemic Way, for example about Inject the compound directly into a solid tumor, often in a depot formulation or in a prolonged formulation Administer release.

In addition, can compound in a targeted drug delivery system, for example, in a tumor-specific antibody coated Liposome, administer. The liposomes are targeted to the tumor be and be selectively absorbed by him. In a preferred Embodiment will be the thyroxine analogues and drugs described herein are administered orally.

Composition / Formulation

The Medicines described here can be used in a way that is is known, e.g. by means of conventional mixing, solution, Granulating, dragee making, pulverizing, emulsifying, encapsulating, Inclusion- or lyophilization process.

drug for use according to the present invention Invention can thus in conventional Way using one or more physiologically acceptable carrier which include excipients and excipients which the processing of the active compounds in preparations containing can be used pharmaceutically. The right Wording is of the chosen one Administration route.

to Injection can the agents of the invention in aqueous Solutions, preferably in physiologically acceptable buffers, such as Hanks solution, Ringer's solution or physiological saline buffer. To transmucosal Administration will be penetrating agents for the barrier to be penetrated are suitable, used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be readily formulated by combining with pharmaceutically acceptable carriers known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing the compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules after adding suitable excipients, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol, cellulose preparations, for example corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as the crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate may be added.

tablet cores are provided with suitable coatings. For this Purpose concentrated sugar solutions gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, Polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic Solvent or Solvent mixtures can contain. Dyes or pigments can the tablets or dragee coatings for labeling or Characterizing different combinations of doses of the active ingredients added become.

pharmaceutical Preparations that can be used orally include capsules, made of gelatin, as well as sealed soft capsules, those made of gelatin and a plasticizer, such as glycerol or sorbitol, are made, a. The capsules can be the effective ingredients in admixture with a filler, such as lactose, binders such as starches, and / or lubricants, such as talc or magnesium stearate, and optionally stabilizers contain. In soft capsules can the active compounds in suitable liquids, such as fatty oils, paraffin oil or liquid polyethylene glycols solved or suspended. Furthermore can Stabilizers are added. All formulations for oral Administration should be in dosages appropriate for one Administration are suitable.

to buccal administration the compositions take the form of tablets or lozenges take in the conventional Are formulated.

to Administration by inhalation, the compounds are for use according to the present Invention expediently in the form of an aerosol spray presentation from pressure packs or a nebulizer using a suitable propellant, e.g. Dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, Carbon dioxide or other suitable gas. in the Case of a pressurized aerosol may be the dosage unit by providing a valve for dispensing a metered Quantity to be determined. Capsules and cartridges of e.g. Gelatin for Use in an inhaler or insufflator can be a powder mixture the compound and a suitable powder base such as lactose or strength, containing formulated.

The Connections can for parenteral administration by injection, e.g. by bolus injection, or continuous infusion can be formulated. Formulations for injection can in dosage units, e.g. in ampoules or in multi-dose containers, with an added one Preservatives are presented. The compositions may be such Forms such as suspensions, solutions or emulsions in oily or aqueous Take and can take vehicles Formulating agents, such as suspending, stabilizing and / or Dispersant, included.

pharmaceutical Formulations for parenteral administration include aqueous solutions of the active ones Compounds in water-soluble Shape. Furthermore can Suspensions of the active compounds as suitable oily injection suspensions getting produced. Suitable lipophilic solvents or vehicles include fatty oils, such as Sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Injection suspensions can Substances that are the viscosity increase the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that contain the solubility increase the connections, to enable the production of highly concentrated solutions.

In another embodiment The active ingredient may be in powder form for preparation with a suitable Vehicle, e.g. sterile pyrogen-free water, before use.

The Connections can also in rectal compositions, such as suppositories or retention enemas, the e.g. conventional Suppository bases, such as cocoa butter or other glycerides, can be formulated.

In addition to The compounds described above may also contain the compounds as a depot preparation be formulated. Such long-acting formulations can by Implantation (for example, subcutaneous or intramuscular) or by intramuscular Be administered injection. So can the connections for example with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or Ion exchange resins or as sparingly soluble derivatives, for example as poorly soluble Salt, to be formulated.

One suitable pharmaceutical carrier for hydrophobic Compounds of the invention is a cosolvent system, the benzyl alcohol, a non-polar surface active A substance, a water-miscible organic polymer and an aqueous phase includes. The cosolvent system can the VPD-cosolvent system be. VPD is a solution from 3% (w / v) benzyl alcohol, 8% (w / v) of the nonpolar surfactant Stoffs polysorbate 80 and 65% (w / v) polyethylene glycol 300, which is made in absolute ethanol to volume. The VPD cosolvent system (VPD: 5W) consists of VPD, 1: 1 with a 5% (w / v) solution Dextrose diluted in water. This cosolvent system dissolves hydrophobic compounds good, and even produces low toxicity after systemic administration. Naturally can the ratios a cosolvent system considerably changed without its solubility and toxicity properties to destroy. Furthermore can the identity the cosolvent components changed be: for example, can other non-polar surface active Substances with low toxicity be used in place of polysorbate 80; the faction area of the polyethylene glycol can be changed become; other biocompatible Polymers can Replace polyethylene glycol, e.g. polyvinylpyrrolidone; and other Sugar or polysaccharides can Replace dextrose.

In another embodiment can other delivery systems for hydrophobic pharmaceutical compounds are used. liposomes and emulsions are well-known examples of delivery vehicles or cost objects for hydrophobic Drugs. Certain organic solvents, such as dimethyl sulfoxide, can also although this is usually at the expense of greater toxicity. In addition, the Compounds using a system with extended Release, such as semipermeable matrices of solid hydrophobic polymers, which contain the therapeutic agent are delivered. Various Types of materials with extended Release are introduced have been and are known to professionals. Capsules with extended Release can dependent on by their chemical nature, the compounds a few weeks up to about Release for 100 days.

The Medicines can also include suitable solid or gel phase carriers or excipients. Examples of such carriers or exclude excipients Calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, Gelatin and polymers such as polyethylene glycols, but are not limited to this.

Other Formulations for administering the thyroxine analogs described herein be suitable, experts will be self-evident and can Example in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, Newest Edition can be found.

Effective dosages

Medicaments suitable for use in the present invention include compositions in which the active ingredients are contained in a therapeutically effective amount. The determination of an effective amount is well within the ability of those skilled in the art, especially in view of the detailed disclosure provided herein. For any compound used in the method of the invention, a therapeutically effective dose may initially be estimated from cell culture assays. For example, a dose can be formulated in animal models to a systemic concentration range to achieve the (ie the concentration of test compound that is lethal to 50% of a cell culture), the I ₅₀ value, as determined in a cell culture, or I ₁₀₀ value, as determined in a cell culture (ie, the concentration of the compound that is lethal to 100% of a cell culture). Such information can be used to more accurately determine the useful doses in humans. Initial dosages may also be formulated by comparing the efficacy of the thyroxine analogs described herein in cell culture assays with the efficacy of known anti-cancer drugs, such as vincristine. In this method, an initial dosage can be obtained by multiplying the ratio of the effective concentrations obtained in a cell culture assay for the thyroxine analog and a known anti-cancer drug with the effective dosage of the known anti-cancer drug. For example, if a thyroxine analog is twice as potent in a cell culture assay as vincristine (ie, the I ₅₀ ^DIME value is one-half the size of the I ₅₀ ^vincristine in the same assay) an initial effective dosage of the thyroxine analog would be half the known dosage for vincristine. Using these initial guidelines, one of ordinary skill in the art could determine an effective dosage in humans.

starting doses can also estimated from in vivo data become. For example, it has been found that 250 mg / kg once Every day 5 days a week 32 days with the nasogastric tube administered the growth of breast cancer xenografts (MDA-MB-231) in nude mice significantly decreased (see example 7.3).

Studies have also shown that serum DIME has a half-life (t _1/2 ) of about 2-2.5 hours and is bioavailable by oral administration to 87% (see Example 7.2). One of ordinary skill in the art could easily optimize administration to humans based on this data.

dosage amount and interval can individually adjusted to plasma levels of effective compound provide sufficient to have a therapeutic effect maintain. Usual patient doses for oral administration are in the range of about 50-2000 mg / kg / day, generally from about 250-1000 mg / kg / day, preferably from about 500-700 mg / kg / day, and most preferably from about 350-550 mg / kg / day. Preferably, therapeutically effective serum levels by administering several doses each day.

In make local administration or selective uptake may be the effective local concentration of the drug not with the plasma concentration be related. A professional will be able to do that therapeutically to optimize effective local dosages without undue experimentation.

The Of course, the amount of the composition administered is determined by the person being treated is the weight of the person, the severity of the disease, the nature of the Administration and the judgment of the prescribing physician.

The Chemotherapy can be repeated intermittently, as long as Tumors are detectable or even if they are undetectable are. Furthermore The therapy may be due to its obvious non-toxicity (below discussed) alone or in combination with other drugs against cancer or other drugs, such as AZT, anti-inflammatory Agents, antibiotics, corticosteroids, vitamins and the like to be provided.

One potential Synergism between the thyroxine analogues described herein and other drugs are expected and predictable. In addition, will also a possible one Synergism expected between a variety of thyroxine analogues and is predictable.

toxicity

The toxicity and therapeutic efficacy of the thyroxine analogs described herein can be determined by standard pharmaceutical techniques in cell cultures or in experimental animals, eg by determining the LD ₅₀ (the dose fatal to 50% of the population) and the ED ₅₀ (the dose therapeutically effective at 50% of the population ). The dose ratio between toxic and therapeutic effect is the therapeutic index and can be expressed as the ratio between LD ₅₀ and ED ₅₀ . Compounds showing high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies may be used in formulating a dosage range that is non-toxic to human use. The dosage of such compounds is preferably within a range of systemic concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. The exact formulation, route of administration and dosage may be chosen by the individual physician in view of the condition of the patient. (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, p. 1).

One of the advantages of using the thyroxine analogues described herein for treating cancer is, among others, their lack of toxicity. For example, it has been found that a daily oral dose of 1 g / kg administered for 12-15 days did not produce adverse effects in nude mice (see Example 7.1). Since the iv serum half-life (t _1/2 ) of DIME is about 2-2.5 hours, repeated daily dosages of the thyroxine analogues described herein are predictable without adverse effects.

The The invention has been described, the following examples are cited to illustrate the subject invention as an example.

Example 1: Compound Synthesis

fourteen Thyroxine analogues have been synthesized, purified and characterized. A summary of the structure of each synthesized Connection and selected Physical data are provided in Table 1 below.

Ref.^a:

Verbindung 6 wurde gemäß Borrows et al., J. Chem. Soc. 1949: S185–S190 hergestellt.

• b: Zersetzungstemperatur

TABLE 1 Synthesized thyroxine analogs

Ref. ^A :

Compound 6 was prepared according to Borrows et al., J. Chem. Soc. 1949: S185-S190 made.

• b: decomposition temperature

1.1 Methyl 3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (Compound 1)

Methyl 3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (Compound 1) was prepared as described in Borrows et al., 1949, "The Synthesis of Thyroxines and Related Substances." Part I. The Preparation of Tyrosine and Some of its Derivatives, and a New Route to Thyroxine, J. Chem. Soc. 1949 (Supp. Issue No. 1): 5185-5190, and recrystallized from 95% ethanol. Melting point: 153-155 ° C.
Mass spectrum: FAB, m / z (relative intensity): 510 (M ⁺ , 100), 479 (4.5), 384 (4.5). High resolution data for the M ⁺ peak: calculated for C ₁₅ H ₁₂ I ₂ O ₄ : 509.882513; found: 509.882960 (deviation = -0.9 ppm).
¹ H-NMR spectrum in DMSO-d ₆ (δ values (ppm) relative to TMS): 3.719 (3H, singlet), 3.876 (3H, singlet), 6.693 (2H, doublet, J = 9.45 Hz, plus fine splitting), 6.845 (2H, doublet, H = 9.36 Hz, plus fine splitting), 8.390 (2H, singlet).

1.2 Methyl 3,5-diiodo-4- (4'-ethoxyphenoxy) benzoate (Compound 2)

Methyl-3,5-diiodo-4- (4'-ethoxyphenoxy) benzoate (Compound 2) was synthesized using the general method of Borrows et al., 1949, "The Synthesis of Thyroxine and Related Substances. Part I. The Preparation of Tyrosine and Some of its Derivatives, and a New Route to Thyroxine, J. Chem. Soc. 1949 (Supp. Issue No. 1): S185-S190, synthesized.

1.2.1 Methyl 3,5-dinitro-4- (4'-ethoxyphenoxy) benzoate

In a 50 ml flask, 4-ethoxyphenol (Aldrich) (1492 mg, 10.8 mmol) was stirred at ambient temperature with 2.0 M aqueous KOH solution (5.50 ml) to give potassium 4-ethoxyphenolate. Methyl 4-chloro-3,5-dinitrobenzoate (Ullmann, 1909, Annalen der Chemie 366: 92-93; reference: Spectrum Chemical Company, Gardena, CA; 2606 mg, 10.0 mmol) was added, the mixture for 1 hour heated to reflux and cooled in an ice bath, whereupon a gummy mass of the product settled. Cold aqueous 1.0 M KOH solution (20 ml) was added and, after continued cooling, the product solidified. The yellow-orange solid was broken, collected on a suction filter, rinsed with water and dried. The material (3.08 g) was recrystallized from hot 95% ethanol (50 ml) to give 2.56 g (70.6% yield) of methyl 3,5-dinitro-4- (4'-ethoxyphenoxy) benzoate were obtained. Melting point: 101-103 ° C.
Mass spectrum (EI): M ⁺ in high resolution: calculated for C ₁₆ H ₁₄ N ₂ O ₈ : 362.075016; found: 362.074793 (deviation = 0.6 ppm).

1.2.2 Methyl 3,5-diiodo-4- (4'-ethoxyphenoxy) benzoate

A portion (724.4 mg, 2.00 mmol) of methyl 3,5-dinitro-4- (4'-ethoxyphenoxy) benzoate was dissolved in glacial acetic acid (50 ml), with 10% palladium on carbon catalyst ( Aldrich) (200 mg) in a Model 4561 Parr mini-reactor, charged with a 43 psi H ₂ atmosphere, and stirred rapidly at ambient temperature until the pressure drop due to reaction ceased (6 minutes, final 16 psi). The mixture was immediately filtered through a celite bed to remove the catalyst and the acetic acid solvent was stripped on a rotary evaporator to give a brown, oily residue which was the crude 3,5-diamine derivative. The crude diamine was dissolved in glacial acetic acid (6.0 mL) and tetrazotised by adding dropwise over 3 minutes to a stirred ice-cold solution of sodium nitrite (345 mg, 5 mmol) in concentrated sulfuric acid (3.5 mL) has been. After stirring for 30 minutes at ice bath temperature, the viscous mixture was pipetted into a rapidly stirred solution of potassium iodide (3.0 g) in distilled water (2.5 ml) at ambient temperature. The dark mixture was stirred for 30 minutes and finally heated to 70 ° C for 5 minutes. The mixture was poured into ethyl acetate (100 ml) and water (50 ml) was added. The biphasic mixture was transferred to a separatory funnel, additional ethyl acetate (50 ml) and water (50 ml) were added and the product was extracted into the ethyl acetate. The organic (ethyl acetate) phase was washed with two additional portions of water (50 ml each) and dried over anhydrous sodium sulfate. Subsequent removal of the ethyl acetate by evaporation afforded a dark, tarry residue.

This crude product was dissolved in acetone (8 ml) and purified by preparative thin layer chromatography plates (five) (Whatman, silica gel, 1000 μm layer, 20 cm x 20 cm, with fluorescent indicator). The plates were developed in n-hexane: ethyl acetate: acetic acid (3: 1: 0.8 v / v / v). The product band (R _f = 0.84), visualized under UV light, was collected from the respective plates, combined and eluted from the silica gel (held in a sintered glass funnel) with ethyl acetate (3 x 50 mL). Removal of the ethyl acetate afforded a cream solid, which was recrystallized from 95% ethanol (10 ml). Yield: a total of 275 mg from two yields of white crystals (26% based on 2 mmol of dinitro precursor). Melting point: 123-125 ° C.
Mass spectrum: EI, m / z (relative intensity): 524 (M ⁺ , 100), 496 (16.7), 310 (9.1), 242 (6.1), 211 (7.6), 155 ( 6.1). High resolution data for the M ⁺ peak: calculated for C ₁₆ H ₁₄ I ₂ O _4: 523.898163; found: 523.898737 (deviation = -1.1 ppm).
¹ H-NMR spectrum in DMSO-d6 (δ values (ppm) relative to TMS): 1.303 (3H, triplet, J = 6.94 Hz), 3.877 (3H, singlet), 3.971 (2H, quartet, J = 6.95 Hz), 6.678 (2H, doublet, J = 8.98 Hz, plus fine splitting), 6.879 (2H, Doublet, J = 9.06 Hz, plus fine splitting), 8.389 (2H, singlet).

1.3 Methyl 3,5-diiodo-4- (4'-n-propoxyphenoxy) benzoate (Compound 3)

Methyl 3,5-diiodo-4- (4'-n-propoxyphenoxy) benzoate (Compound 3) was prepared as described in Example 1.2. The dinitro precursor was synthesized by treating an aqueous solution of potassium 4-n-propoxyphenolate (made from commercial 4-n-propoxyphenol) with methyl 4-chloro-3,5-dinitrobenzoate. The dinitro product was reduced by H ₂ / Pd (C) to the diamine derivative, which was then tetrazotised with NaNO ₂ / H ₂ SO ₄ and converted to the diiodo product by reaction with potassium iodide (Sandmeyer reaction). Purification was by preparative TLC and crystallization.

1.4 Methyl 3,5-diiodo-4- (4'-n-butoxyphenoxy) benzoate (Compound 4)

Methyl 3,5-diiodo-4- (4'-n-butoxyphenoxy) benzoate (Compound 4) was prepared as described in Example 1.2. The dinitro precursor was synthesized by treating an aqueous solution of potassium 4-n-butoxy phenolate (made from commercial 4-n-butoxyphenol) with methyl 4-chloro-3,5-dinitrobenzoate. The dinitro product was reduced by H ₂ / Pd (C) to the diamine derivative, which was then tetrazotised with NaNO ₂ / H ₂ SO ₄ and converted to the diiodo product by reaction with potassium iodide (Sandmeyer reaction). Purification was by preparative TLC and crystallization.

1.5 ethyl 3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (Compound 5)

Ethyl 3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (Compound 5) was synthesized via 3,5-diiodo-4- (4'-methoxyphenoxy) benzoyl chloride, the latter in Borrows et al., 1949 , 1. Chem. Soc. 1949: S185-S190 has been described. Thus, 3,5-diiodo-4- (4'-methoxyphenoxy) benzoic acid (99.2 mg, 0.200 mmol) in a 10 ml flask was converted to 3,5-diiodo-4- (4'-methoxyphenoxy) benzoyl chloride. After removing the excess thionyl chloride under vacuum, anhydrous ethanol (5.0 ml) was added with stirring and the mixture was heated to 70 ° C for 5 minutes. Excess ethanol was removed and the dry residue was dissolved in hot 95% ethanol (4.0 ml), from which the product ester crystallized in the refrigerator (3 ° C). Yield: 55.8 mg (53%) of tan-colored crystals.
Melting point: 96-98 ° C.
Mass spectrum (EI): High resolution data for the M ⁺ peak: calculated for C ₁₆ H ₁₄ I ₂ O _4: 523.898163; found: 523.898202 (deviation = -0.1 ppm).
¹ H-NMR spectrum in DMSO-d ₆ (δ values (ppm) relative to TMS): 1.336 (3H, triplet, J = 7.19 Hz), 3.717 (3H, singlet), 4.336 (2H, quartet, J = 7.06 Hz), 6.695 (2H, doublet, J = 9.34 Hz, plus fine splitting), 6.895 (2H, doublet, J = 9.20, plus fine splitting), 8.389 (2H, singlet).

1.6 3,5-Diiodo-4- (4'-methoxyphenoxy) benzoic acid (Compound 6)

3,5-diiodo-4- (4'-methoxyphenoxy) benzoic acid (Compound 6) was synthesized as described in Borrows et al., 1949, J. Chem. Soc. 1949: S185-S190 is described.

1.7 3,5-Diiodo-4- (4'-methoxyphenoxy) benzamide (Compound 7)

3,5-Diiodo-4- (4'-methoxyphenoxy) benzamide (Compound 7) was synthesized by amidating Compound 1. In a 125 ml flask, methyl 3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (compound 1) (100 mg, 0.196 mmol) in anhydrous methanol (60 ml) was dissolved. Anhydrous ammonia gas was bubbled into the solution for 5 minutes at a moderate rate at ambient temperature. After standing for one hour in the sealed flask, the ammonia gas treatment was repeated (5 minutes) and the mixture allowed to stand in the sealed flask for 48 hours. The methanol / ammonia was removed by rotary evaporation, the dry residue dissolved in methanol: water (7: 3 v / v) (30 ml) and crystallized in the refrigerator (3 ° C). Yield: 58.3 mg (60% yield) of yellow-brown colored crystals. Melting point: 207-209 ° C.
Mass Spectrum (FAB): High-resolution data for the M ⁺ peak: calculated for C ₁₄ H ₁₁ I ₂ NO _3: 494.882847; found: 494.881880 (deviation = 2.0 ppm).
¹ H-NMR spectrum DMSO-d6 (δ values (ppm) relative to TMS): 3.716 (3H, singlet), 6.682 (2H, doublet, J = 8.93 Hz), 6.895 (2H, doublet, J = 8.99 Hz), 7.528 (1H, singlet), 8.113 (1H, singlet), 8.402 (2H, singlet).

1.8 3,5-Diiodo-4- (4'-methoxyphenoxy) -N-methylbenzamide (Compound 8)

3,5-diiodo-4- (4'-methoxyphenoxy) -N-methylbenzamide (Compound 8) was over 3,5-diiodo-4- (4'-methoxyphenoxy) benzoyl chloride (see Example 1.5). The acid chloride was treated with excess methylamine reacted in tetrahydrofuran at ambient temperature (1 hour), filtered to remove methylamine hydrochloride precipitate, the solvent evaporated and the product crystallized from 95% ethanol.

1.9 3,5-Diiodo-4- (4'-methoxyphenoxy) -N, N-dimethylbenzamide (Compound 9)

3,5-diiodo-4- (4'-methoxyphenoxy) -N, N-dimethylbenzamide (Compound 9) was over 3,5-diiodo-4- (4'-methoxyphenoxy) benzoyl chloride (see Example 1.5). The acid chloride was treated with excess methylamine reacted in tetrahydrofuran at ambient temperature (1 hour), filtered to remove methylamine hydrochloride precipitate, the solvent evaporated and the product crystallized from absolute ethanol.

1.10 Methyl 3,5-diiodo-4- (4'-hydroxyphenoxy) benzoate (Compound 10)

Methyl-3,5-diiodo-4- (4'-hydroxyphenoxy) benzoate (Compound 10) was prepared as described in Example 1.2 is. The dinitro precursor was prepared by reacting 4-chloro-3,5-dinitrobenzoate with Hydroquinone in pyridine solution as described in Borrows et al., 1949, "The Synthesis of Thyroxine Related Substances. Part II. The Preparation of Dinitrodiphenyl Ethers, J. Chem. Soc. 1949 (Supp. Issue No. 1): S190-S199 is described.

1.11 Methyl 3,5-diiodo-4-phenoxybenzoate (Compound 11)

Methyl 3,5-diiodo-4-phenoxybenzoate (Compound 11) was prepared as described in Example 1.2. The dinitro precursor was synthesized by treating an aqueous solution of potassium phenoxide (made from commercial phenol) with methyl 4-chloro-3,5-dinitrobenzoate. The dinitro product was reduced by H ₂ / Pd (C) to the diamine derivative, which was then tetrazotised with NaNO ₂ / H ₂ SO ₄ and converted to the diiodo product by reaction with potassium iodide (Sandmeyer reaction). Purification was by preparative TLC and crystallization.

1.12 Methyl 3,5-diiodo-4- (4'-iodophenoxy) benzoate (Compound 12)

Methyl 3,5-diiodo-4- (4'-iodophenoxy) benzoate (Compound 12) was synthesized as described in Example 1.2. Since the iodine substituent in the dinitro precursor itself is labile to reduction by H ₂ / Pd (C), the iododinitro precursor was reduced to iododiamine with iron powder in acetic acid / 95% ethanol (see, eg, Gemmill et al., 1956, 3-iodo , 3,3'-diiodo and 3,3'-diiodo-5-bromothyroxine, J. Am. Chem. Soc. 78: 2434-2436). The iodo-diamine was then tetrazotised and converted to the triiodo product using the Sandmeyer reaction. After purification by preparative TLC, the product (mp 139-141 ° C) was crystallized from ethanol.
Mass spectrum (EI): high resolution data for the M ⁺ peak: calculated for C ₁₄ H ₉ O ₃ I ₃ : 605.768600; found: 605.767839 (deviation = 1.3 ppm).
¹ H-NMR spectrum DMSO-d6 (δ values (ppm) relative to TMS): 3.879 (3H, singlet), 6.628 (2H, doublet, J = 8.97 Hz plus fine splitting), 7.670 (2H, doublet, J = 9.12 Hz plus fine splitting), 8.396 (2H, singlet).

1.13 Methyl 3,5-diiodo-4- (3'-methoxyphenoxy) benzoate (Compound 13)

Methyl 3,5-diiodo-4- (3'-methoxyphenoxy) benzoate (Compound 13) was synthesized as described in Example 1.2. The dinitro precursor was synthesized by treating an aqueous solution of potassium 3-methoxyphenolate (made from commercial 3-methoxyphenol) with methyl 4-chloro-3,5-dinitrobenzoate. The dinitro product was reduced by H ₂ / Pd (C) to the diamine derivative, which was then tetrazotised with NaNO ₂ / H ₂ SO ₄ and converted to the diiodo product by reaction with potassium iodide (Sandmeyer reaction). Purification was by preparative TLC and crystallization.

1.14 Methyl 3-5-diiodo-4- (2'-chloro-4'-methoxyphenoxy) benzoate (Compound 14)

Methyl 3,5-diiodo-4- (2'-chloro-4'-methoxyphenoxy) benzoate (Compound 14) was prepared by the general method described in Example 1.2 but with an alternative method of reducing Dini trovorstufe synthesized.

1.14.1 Methyl 3,5-dinitro-4- (2'-chloro-4'-methoxyphenoxy) benzoate

The dinitro precursor was prepared by reacting 2-chloro-4-methoxyphenol (Aldrich Chemical Co., Milwaukee, WI) as potassium 2-chloro-4-methoxyphenolate with methyl 4-chloro-3,5-dinitrobenzoate as described in U.S. Pat Example 1.2.1 is described. The methyl 3,5-dinitro-4- (2'-chloro-4'-methoxyphenoxy) benzoate product (66% yield) was crystallized from ethanol to give orange crystals. Melting point: 116-119 ° C.
Mass spectrum (EI): M ⁺ at high resolution: calculated for C ₁₅ H ₁₁ ClN ₂ O ₈ : 382.020393; found: 382,020187 (deviation = 0.5 ppm).

1.14.2 Methyl 3,5-diiodo-4- (2'-chloro-4'-methoxyphenoxy) benzoate

Since the 2'-chloro substituent in the dinitro precursor is labile to reduction by H ₂ / Pd (C), the precursor was reduced to 2'-chlorodiamine with iron powder in acetic acid / 95% ethanol, similar to Example 1.12. Thus, in a 250 ml flask, methyl 3,5-dinitro-4- (2'-chloro-4'-methoxyphenoxy) benzoate (765.5 mg, 2.00 mmol) in glacial acetic acid (35 ml) and 95% strength Dissolved ethanol (35 ml), the solution heated to 70 ° C and iron powder (2.00 g) was added. The mixture was vigorously vortexed in a heating bath (70 ° C). After vortexing for 3 minutes, the mixture developed a brown color. The vortexing was continued for 35 min. At 70 ° C. The mixture was then transferred to a separatory funnel, water (250 ml) and ethyl acetate (250 ml) added, the product was extracted into the ethyl acetate phase and the ethyl acetate phase allowed to separate from the aqueous phase (3 hours). The extract was dried over anhydrous Na ₂ SO ₄ , filtered and the ethyl acetate removed by rotary evaporation to give the crude 3,5-diaminoproduct which solidified.

The crude diaminoproduct was immediately dissolved in glacial acetic acid (6.0 ml), tetrazotised and converted to the methyl 3,5-diiodo-4- (2'-chloro-4'-methoxyphenoxy) benzoate by the Sandmeyer reaction as described in US Pat Example 1.2 is described. After purification by preparative thin layer chromatography (R _f = 0.70), as described in Example 1.2, the product was crystallized from 95% ethanol (250.8 mg cream crystals, 23% yield). Melting point: 132-134 ° C.
Mass spectrum: EI, m / z (relative intensity): 546 (34), 545 (16), 544 (M ⁺ , 100), 418 (6), 382 (6). High resolution data for the M ⁺ peak: calculated for C ₁₅ H ₁₁ ClI ₂ O ₄ : 543.843541; found: 543,843424 (deviation = 0.2 ppm).
¹ H-NMR spectrum in DMSO-d ₆ (δ values (ppm) relative to TMS): 3.747 (3H, singlet), 3.881 (3H, singlet), 6.328 (1H, doublet, J = 8.97 Hz) , 6.780 (1H, doublet of doublets, J = 9.10 Hz and J = 2.95 Hz), 7.195 (1H, doublet, J = 3.02 Hz), 8.400 (2H, singlet).

1.15 Other connections

additional Thyroxine analogs described herein may be prepared using the above synthesized synthesized from corresponding starting materials As it is readily apparent to those skilled in organic chemistry will be. additional Guidance can be found in the art, especially in Borrows et al., 1949, "The Synthesis of Thyroxine and Related Substances. Part I. The Preparation of Tyrosine and Some of its Derivatives, and a New Route to Thyroxine, J. Chem. Soc. 1949 (Supp. Issue No. 1): S185-S190; Borrows et al., 1949, "The Synthesis of Thyroxine and Related Substances. Part II. Preparation of Dinitrophenyl Ethers, "J. Chem. Soc. 1949 (Supp. Issue No. 1): S190-S199; Clayton et al., 1951, "The Synthesis of Thyroxine and Related Substances. Part VIII. The Preparation of Some halogeno and nitro diphenyl ethers, J. Chem Soc., 1951: 2467-2473; Gemmill et al., 1956, "3-iodo, 3,3'-diiodo and 3,3'-diiodo-5-bromothyroxine, "J. Am. Chem. Soc. 78: 2434-2436; Meltzer et al., 1957, "Thyroxine Analogs," J. Org. Chem. 22: 1577-1581; Crowder et al., 1958, "Bisbenzylisoquinolines. Part Π. The Synthesis of 5- (2-aminoethyl) -4'-carboxy-2,3-dimethoxydiphenyl Ether, J. Chem. Soc. 1958: 2142-2149; Jorgensen, 1978, "Thyroid Hormones and Analogues, I. Synthesis, Physical Properties and Theoretical Calculations "In: Hormonal Proteins and Peptides Vol. VI, pp. 57-105, C.H. Li, eds., Academic Press, NY (and references cited therein); and Jorgensen, 1978, "Thyroid Hormones and Analogues, II. Structure-Activity Relationships, "In: Hormonal Proteins and Peptides, Vol. VI, pp. 107-204, C.H. Li, eds., Academic Press, NY (and references cited therein).

EXAMPLE 2: ACTIVATION PURIFIED PROTEIN PHOSPHATASE 2A

The Compounds 1 and 3, as indicated in Table 1, were tested for protein phosphatase 2A activation.

2.1 Preparation of ³² P-labeled histone H1 substrate

Histone H1 was phosphorylated with protein kinase C (Upstate Biotechnology, Inc., Lake Placid, NY) in a volume of 100 μl according to standard protocols, in another embodiment histone H1 was phosphorylated with p34cdc2 kinase derived from rapidly multiplying Mytilus Edulis embryo in the Stage 4 - 8 was purified after isolation with the P ¹³ -Suc-agarose technique (Smythe and Newport, 1992, "Coupling of Mitosis to the Completion of S Phase in Xenopus Occurs via Modulation of the Tyrosine Kinase that Phosphorylates p34cdc2," Cell 68: 787-797).

2.2 Phosphatase Assay

125 ng of purified protein phosphatase 2A (Upstate Biotechnology, Inc., Lake Placid, NY; Usui et al., 1983, J. Biol. Chem. 258: 10455-10463) was mixed with thyroxine analog (50 μM) in buffer (20 mM MOPS or Tris, pH 7.5, 1 mM MgCl ₂ , 60 μM β-mercaptoethanol) at 23 ° C for 10 min. Preincubated. The total reaction volume was 20 μl. ³² P-labeled histone H1 substrate (10 μg, 10 ⁵ cpm) was added and the dephosphorylation reaction allowed to proceed for 5 min at 23 ° C, and after that time, the reaction was quenched by the addition of 2 μl of Laemmli buffer. An identical reaction containing 125 ng of untreated protein phosphatase 2A was run as a control.

Phosphorylated and dephosphorylated histone H1 were separated by gel electrophoresis (12% SDS-PAGE), bands containing phosphorylated histone H1 were excised and assayed for ³² P activity by a scintillation counter.

2.3 Results

Angegebene Werte sind der Mittelwert von drei Proben.The rate of dephosphorylation in 5 min. Is an indication of the initial rate (V _init ) of the dephosphorylation reaction. V _init for compounds 1 and 3 is provided in Table 2 below. TABLE 2 Activation of protein phosphatase 2A

Stated values are the mean of three samples.

These results show that a short (10 min) preincubation of protein phosphatase 2A with DIME (Compound 1) V _init more than doubles histone H1 dephosphorylation. The 4'-propoxy homologue did not activate protein phosphatase 2A. These data indicate that even minor changes in the ends of the DIME pharmacophore structure (ie, the methoxy and methyl ester groups) significantly affect activity. These data correlate strongly with protein phosphatase 2A activation observed in in vitro malignant cell assays (see Example 3) and in vivo antitumour activity as observed in mice (see Examples 4 and 6).

EXAMPLE 3: ACTIVATION PROTEIN PHOSPHATASE A2 IN TUMOR CELL CULTURES

Pi

= Anorganisches Phosphat

Compounds 1-13 were tested for protein phosphatase 2A activation in malignant tumor cell cultures in vitro according to the protocol described in Bauer et al., 1996, "Modification of Growth Related Enzymatic Pa Thways and Apparent Loss of Tumorigenicity of a Ras-Transformed Bovine Endothelial Cell Line by Treatment with 5-iodo-6-amino-1,2-benzopyrone (INH ₂ BP), "International J. of Oncology 8: 239-252 is described The results for activation by DIME (Compound 1) are tabulated in Table 3. All other compounds were ineffective TABLE 3 Effect of DIME on the phosphatase activity of E-ras 20-cell nuclear extract

pi

= Inorganic phosphate

These Results show that 50 μM DIME protein phosphatase 2A in both E-ras transformed bovine endothelial cells and at least doubled in DU-145 cells.

EXAMPLE 4: CELL DAMAGING EFFECT ON HUMAN CANCER CELLS

The cytocidal activity of Compounds 1-13 was evaluated in vitro at seven tested human cancer cell lines. DIME (connection 1) was maximum effective, wherein the ethoxy derivative (compound 2) 25-30% of had maximum effectiveness.

4.1 experimental protocol

Seven human cancer cell lines were obtained from the American Tissue Culture Collection (Rockville, MD) and maintained in the recommended growth media. The cells were seeded in wells (2 cm ² ) at a density of 2 × 10 ⁴ cells / cm ² . Various concentrations of compounds 1-13 were added to the media at the time of sowing.

The cultures were incubated for 72 hours at 37 ° C (5% CO ₂ atmosphere). After incubation, the cells were detached with trypsin and counted in a hemocytometer.

4.2 Results

DIME (Compound 1) was maximally effective, with the ethoxy analog (Compound 2) 25-30% the maximum effectiveness. All other tested analogs (Connections 3-13) were completely ineffective.

The experimental results for DIME (Compound 1) are tabulated in Table 4 below. I ₁₀₀ denotes the concentration at which no viable cells remained; I _{50 is} the concentration at which 50% viable cells were left compared to a control.

TABLE 4 Effect of DIME on various human cancer cell lines

EXAMPLE 5: INHIBITION OF THE TUMOR CELL GROWTH

The Compounds 1 and 14 were designed to inhibit the growth of MDA-MD-231 Cancer cells tested. Compound 14 showed about 25% inhibitory potency in comparison to DIME (connection 1).

5.1 trial protocol

human MDA-MD-231 cancer cells were obtained from the American Tissue Culture Collection (Rockville, MD) and in the recommended growth media maintained. Cells were in the presence of various concentrations of compounds 1 and 14 for 3 days at 37 ° C.

5.2 Results

The Test results are tabulated in Table 5 below.

TABLE 5 Growth rate of MDA-MD-231 cells

The 2'-Chloro analogue of DIME (compound 14) showed about 25% inhibitory potency compared to DIME (connection 1).

EXAMPLE 6: LOSS OF THE TUMOR-GENERATING EFFECT OF E-RAS TRANSFORMED BOVINE ENDHELIAL CELLS

The morphological action of DIME (Compound 1) was in a highly tumorigenic E-ras transformed Bovine endothelial cell line tested.

6.1 Experimental protocol

E-ras-transformed bovine endothelial cells (Bauer et al., 1996, Intl. J. Oncology 8: 239-252) were exposed to 10 μM DIME for 3 days. The DIME-treated cells (10 ⁵ or 10 ⁶ cells / 100 μl) were injected subcutaneously into nude mice and tumor development was followed for 25 days.

6.2 Results

As it is in 1 For example, the exposure of 10 μM DIME to E-ras-transformed endothelial cells for a period of 3 days induced significant microkernel formation that coincides with a loss of tumorigenicity. As it is in 2 1, animals exposed to untreated cells showed tumor growth (upper curves), succumbing to the tumors after 25 days. Exposure of 10 μM DIME to cells prior to injection almost completely abolished tumorigenicity (lower curves). Tumors did not appear even after 3 months without in vivo drug treatment.

EXAMPLE 7: IN VIVO EXPERIMENTS

The following examples demonstrate the nontoxicity, bioavailability, serum half life (t _1/2 ) and in vivo efficacy of DIME in treating human breast cancer xenografts in mice.

7.1 Toxicity

Ten nude mice were given a daily oral dose of ¹⁴ C-labeled DIME (Compound 1) (1.0 g / kg, 0.1 ml in corn oil) over a period of 12-15 days. No adverse effects were observed in any of the mice throughout the treatment period.

7.2 Serum Half Life (t _1/2 ) and Bioavailability Mice were orally dosed with 126 mg / kg ¹⁴ C-labeled DIME (Compound 1). After dose administration, blood sampling times were 15 and 30 minutes and 1, 2, 4, 6, 8 and 24 hours. Aliquots (50 μl) of blood were assayed in a liquid scintillation counter and the data expressed as microgram equivalents per ml. Blood level data were collected by the RSTRIP method (Micromath, Salt Lake City, UT).

Parallel groups of mice were intravenously dosed with 24.5 mg / kg ¹⁴ C-labeled DIME and blood sampling times were 10, 20 and 30 minutes and 1, 2, 4, 6 and 8 hours.

7.2.1 Results

Blood serum levels of ¹⁴ C-labeled DIME (mg-eq / ml) are determined in 3 illustrated. The area under the blood concentration-time curve was 665.28 μg-hr / ml for the oral route (data shown by circles) and 156 μg-hr / ml for the intravenous route (data represented by squares). The bioavailability of orally administered DIME was calculated from these data to be 83% using a standard ratio x dose method. The DIME half-life (t _1/2 ) was about 2-2.5 hours.

7.3 In vivo efficacy

The ability human tumors, as xenografts in athymic mice (e.g. Nude mice) to grow provides a useful in vivo model for study the biological reaction. Therapies for human tumors ready. Since the first successful xenotransplantation of human tumors in athymic mice (Rygaard and Povlsen, 1969, Acta Pathol. Microbiol. Scand. 77: 758-760) many different human tumor cell lines (e.g., breast, lungs, Genitourinary, gastrointestinal, head and neck, glioblastoma, bone and malignant melanomas) are successfully transplanted and grown in nude mice been left. Human breast tumor cell lines, the MCF-7, ZR75-1 and MDA-MB-231 as subcutaneous transplants in nude mice (Warri et al., 1991, Intl. J. Cancer 49: 616-23; Ozzello & Sordat, 1980, "Behavior of Tumors Produced by Transplantation of Human Mammary Cell Lines in Athymic Nude Mice, "Eur. J. Cancer 16: 553-559; Osbourne et al., 1985, Cancer Res. 45: 584-590; Siebert et al., 1983, Cancer Res. 43: 2223-2239).

This Experiment shows inhibition of MDA-MB-231 xenografts Nude mice.

7.3.1 Experimental protocol

MDA-MB-231 (human breast cancer) cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in the recommended growth media. Twenty nude mice were each inoculated subcutaneously with MDA-MB-231 cells (10 ⁶ cells / 100 μl). A group of ten mice were given DIME by gavage (250 mg / kg, 10 ml / kg in corn oil) once a day, 5 days a week for a total of 32 days. The other (control) group of ten mice were given vehicle only according to the same dosing regimen. Tumors were measured twice a week using a Vernier probe and the average tumor volume was determined at each time point. Comparisons between the groups were performed using a two-sided untargeted t-test and the results were analyzed using variance analysis.

7.3.2 Results

The average tumor mass on days 14, 21, 28 and 32 after Inoculation is for treated and untreated mice tabulated in Table 5.

• ^aSEM = Standardabweichung des Mittelwerts

TABLE 6 MDA-MB-231 Tumor volume after DIME treatment

• ^a SEM = standard deviation of the mean

These Data show that DIME significantly reduces malignant tumor growth causes, even under a non-optimized treatment protocol.

7.4 In vivo efficacy

Other Thyroxine analogs described herein are tested as described above is. According to these Assays are expected to show the analogous efficacy.

EXAMPLE 8: Formulations

The The following examples are examples of non-limiting formulations for administering the thyroxine analogues of the invention to mammalian, in particular Human patients ready. Although the examples contain formulations of DIME show, it goes without saying that any of the thyroxine analogs described herein formulated can be as provided in the following examples.

8.1 tablet formulation

tablets, each containing 60 mg of active ingredient are composed as follows:

Of the Active ingredient, starch and cellulose are passed through a No. 45 mesh (US) sieve and mixed thoroughly. The polyvinylpyrrolidone solution is mixed with the resulting powders, which are then passed through a Sieve with No. 14 mesh (US) to be passed. The granules are added Dried 50 ° -60 ° C. and passed through a No. 18 mesh (US) sieve. The sodium carboxymethyl starch, magnesium stearate and talc previously passed through a No. 60 mesh (US) sieve then added to the granules, after mixing by a tabletting machine is compressed, with each 150 mg weighing tablets become.

tablets can from the components listed in Table 1 by wet granulation, followed by pressing.

8.2 gelatin capsules

Hard gelatin capsules are made using the following ingredients:

The The above ingredients are mixed and mixed in 460 mg quantities Filled hard gelatin capsules.

8.3 Aerosol solution

A aerosol solution is made containing the following ingredients:

Of the Active substance is mixed with ethanol and the mixture is part of Propellant gas 22 was added, cooled to -30 ° C and transferred to a filling device. The required amount is then fed to a stainless steel container and with the The rest of the propellant diluted. The valve units are then installed on the container.

8.4 suppositories

Each 225 mg of active ingredient containing suppositories become like this produced:

Of the Active ingredient is passed through a sieve with No. 60 mesh (US) and in the saturated fatty acid glycerides previously suspended using the minimum necessary Heat melted become. The mixture is then placed in a suppository mold with a nominal capacity poured 2 g and allowed to cool.

8.5 suspensions

Each 50 mg drug per 5 ml dose containing suspensions as follows produced:

Of the Active ingredient is passed through a sieve with No. 45 mesh (US) and mixed with the sodium carboxymethyl cellulose and the syrup, wherein a smooth paste is formed. The benzoic acid solution, taste and some dye are diluted with some of the water and added with stirring. Enough Water is then added to make the required volume.

EXAMPLE 9

Substitution of an H atom in position 5 of the 6-amino-1,2-benzopyrene with an iodine atom significantly increased the PADPRT-inhibitory activity, the anti-HIV activity and the antitumor activity of the parent compound. Cole et al., 1991 "Inhibtion of HIV-1 IIIb Replication in AA-2 Cells in Culture by Two Ligands of Poly (ADP-Ribose) Polymerase: 6-Amino-1,2-benzopyrone and 5-iodo-6-amino -l, 2-benzopyrone "Biochem. Biophys. Res. Commun. 180: 504-514; Bauer et al., 1996, "Modification of Growth Related Enzymatic Pathways and Apparent Loss of Tumorigenicity of a Ras-Transformed Bovine Endothelial Cell Line by Treatment with 5-iodo-6-amino-1,2-benzopyrone (INH ₂ BP)" Intl. J. Oncol. 8: 239-252. The question was raised as to whether increasing the number of iodine substitutions in certain aromatic molecules may or may not alter their molecular pharmacological properties. To address this question, we first analyzed the cellular action of known diiodo compounds, such as thyroid hormone analogues.

It has been found that metabolic or metamorphogenic effects of thyroid hormone analogues depend on their chemical structure. E. Jorgensen 1978, "Thyroid Hormones and Analogs II. Structure-Activity Relationships," In: Hormonal Protein and Peptides Vol. VI, CH Li (ed.), Academic Press, New York, pp. 108-203. The hormonally inactive methyl 3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME) was first synthesized in 1949 (Borrows et al., 1949, The Synthesis of Thyroxines and Related Substances, Part I. The Preparation of Tyrosine and Some of its Derivatives, and a New Route to Thyroxine ", J. Chem. Soc. Suppl., Issue No. 1: S185-S190), but no significant metabolic or metamorphic effect of this substance has been reported, Money et al , 1958, "The Effect of Change in Chemical Structure of Some Thyroxine Analogues on the Metamorphosis of Rana pipiens Tadpoles", Endocrinology 63: 20-28; Stasili et al., 1959, "Antigoitrogenic and Calorigenic Activities of Thyroxine Analogues in Rats", Endocrinology 64: 62-82; Money et al., 1959, "The Effect of Various Thyroxine Analogues on Suppression of ¹³¹ I Uptake by the Rat Thyroid", Endocrinology 64: 123-125; Kumaoka et al., 1960, "The Effect of Thyroxine Analogues on a Transplantable Mouse Pituitary Tumor", Endocrinology 66: 32-38; Grinberg et al., 1962. "Studies with Mouse Pituitary Thyrotropic Tumors. V. Effect of Various Thyroxine Analogs on Growth and Secretion", Cancer Res. 22: 835-841. In a work begun earlier by the inventors, DIME has been shown to be a potential tumoricidal agent both in cell cultures and in vivo. Kun et al., 1996, "Induction of Tumor Apoptosis by Methyl 3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME)", abstract no. 102, Int. J. Oncol. 9: Supplement 829; Zhen et al., 1997, "Induction of Metaphase Block and Endoreduplication in Human Cancer Cells by 3,5-Diiodo-4- (4'-methoxyphenoxy) benzoate (DIME)". Summary, Amer. Assoc. Cancer Res., Symposium on Cell Signaling and Cancer. The synthesis and testing of structurally homologs and analogs of DIME, which differ only in side chain substitutions, as described in co-pending US patent application Ser. No. 08 / 655,267, filed June 4, 1996, "Method of Treating Malignant Tumors with Thyroxine Analogs Having No Significant Hormonal Activity", outlined the structural specificity of DIME for tumoricidal action.

The following work shows the structure-effect comparison of DIME and 17 of its analogs, and describes the tumoricidal action of DIME even on a cellular Level. Drug metabolism and cellular uptake assays with DIME showed the reasons for the Lack of its toxicity in animals in vivo. The cytometric analysis of the mode of action from DIME and biochemical mechanisms, the objects are more continuous Investigations.

substituted Phenols for the synthesis of compounds 1-4 and 10-18 in Table 1 were obtained from Aldrich Chemical Co. (Milwaukee, WI, USA). receive. Methyl 4-chloro-3,5-dinitrobenzoate, F. Ullmann, 1909, "The 4-chloro-3,5-dinitrobenzoic acid", Annalen der Chemie 36: 92-93, was prepared from 4-chloro-3,5-dinitrobenzoic acid (Aldrich).

9.1 General Synthesis

Each substituted phenol (as its potassium phenolate) was reacted with methyl-4-chloro-3,5-dinitrobenzoate to give the methyl 3,5-dinitro-4- (substituted phenoxy) benzoate, which was then converted to the corresponding 3,5 Diamine was reduced and converted by the Sandmeyer reaction to the target 3,5-diiodo compound. Kun et al., 1996, "Induction of Tumor Apoptosis by Methyl 3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME)", abstract no. 102, Int. J. Oncol. 9: Supplement 829. Generally, the reduction was by catalytic hydrogenation, but for cases where R ₁ or R _{3 is} a halogen atom (Compounds 12 and 14), reduction was by iron powder in acetic acid / ethanol to avoid dehalogenation. Kun et al., 1996, "Induction of Tumor Apoptosis by Methyl 3,5-diiodo-4- (4'-methoxyphenoxy) Benzoate (DIME)", Abstract No. 102, Int. J. Oncol. 9: Supplement 829. The purification was generally carried out by preparative thin-layer chromatography and crystallization. For compounds where R ₂ in Table 7 is other than methoxy (ie, compounds 5-9), additional synthetic reactions were used. Compound 6 was prepared by base hydrolysis of Compound 1. Borrows et al., 1949, "The Synthesis of Thyroxines and Related Substances. Part I. The Preparation of Tyrosine and Some of Its Derivatives, and a New Route to Thyroxine", J. Chem. Soc. Suppl. Issue # 1: S185-S190. Compounds 5, 8, and 9 were prepared by reacting the acid chloride of 6, Borrows et al., 1949, "The Synthesis of Thyroxines and Related Substances." Part I. The Preparation of Tyrosine and Some of its Derivatives, and a New Route to Thyroxine. , J. Chem. Soc. Suppl. Issue No. 1: S185-S190, made with anhydrous ethanol, methylamine, and dimethylamine, respectively. Kun et al., 1996, "Induction of Tumor Apoptosis by Methyl 3,5-diiodo-4- (4'-methoxyphenoxy) Benzoate (DIME)", abstract Nr, 102, Int. J. Oncol. 9: Supplement 829. Compound 7 was obtained by reacting 1 with ammonia in anhydrous methanol. All compounds except the known carboxylic acid 6 were characterized by melting point and high resolution mass spectrometry (Table 7). ¹ H NMR spectra were measured for compounds 1-14 and were satisfactory in all cases.

9.2 Synthesis of the compound 1

The Synthesis of Compound 1 (DIME) was carried out as previously described by Borrows et al., 1949, "The Synthesis of Thyroxine and Related Substances. Part I. The Preparation of Tyrosine and Some of its Derivatives, and a New Route to Thyroxine ", J. Chem. Soc. Suppl. Issue # 1: S185-S190 is described, mp 153-155 ° C. Basic Spectral measurements earlier not for these compounds are given are as follows.

UV absorption spectrum in ethanol, τ max (ε): 289 nm (4.20 × 10 ³ ), 232 nm (3.08 × 10 ⁴ ), 213 nm (2.48 × 10 ⁴ ).
Mass spectrum: FAB, m / z (relative intensity): 510 (M ⁺ , 100), 479 (4.5), 384 (4.5). High resolution data for the M ⁺ peak: calculated for C ₁₅ H ₁₂ I ₂ O ₄ : 509.882513; found: 509.882960 (deviation = -0.9 ppm).
¹ H-NMR spectrum in DMSO-d ₆ (δ values (ppm) relative to TMS): 3.719 (3H, singlet), 3.876 (3H, singlet), 6.693 (2H, doublet, J = 9.45 Hz, plus fine splitting), 6.845 (2H, doublet, J = 9.36 Hz, plus fine splitting), 8.390 (2H, singlet).

9.3 Synthesis of the compound 7

Synthesis of Compound 7 (3,5-diiodo-4- (4'-methoxyphenoxy) benzamide) was prepared by bubbling ammonia into a solution of Compound 1 (100 mg, 0.196 mmol) in anhydrous methanol (60 mL) for 5 min. reached at ambient temperature. After standing for one hour in a sealed flask, the mixture was again treated with ammonia and allowed to stand closed for 48 hours. The methanol / ammonia was removed by rotary evaporation, the dry residue dissolved in warm methanol: water (7: 3 v / v) (30 ml) and crystallized in the refrigerator (3 ° C). Yield: 58.3 mg (60%) of tan-colored crystals, mp 207-209 ° C.
Mass Spectrum (FAB): High-resolution data for the M ⁺ peak: calculated for C ₁₄ H ₁₁ I ₂ NO _3: 494.882847; found: 494.881880 (deviation = 2.0 ppm).
¹ H-NMR spectrum in DMSO-d ₆ (δ values; (ppm) relative to TMS): 3.716 (3H, singlet), 6.682 (2H, doublet, J = 8.93 Hz, plus fine resolution), 6.895 ( 2H, doublet, J = 8.99 Hz, plus fine resolution), 7.528 (1H, singlet), 8.113 (1H, singlet), 8.402 (2H, singlet).

9.4 cell cultures

E-ras 20 cells were cultured as indicated, Bauer et al., 1996, "Modification of Growth Related Enzymatic Pathways and Apparent Loss of Tumorigenicity of a ras-Transformed Bovine Endothelial Cell Line by Treatment with 5-iodo-6. amino-1, 2-benzopyrone (INH ₂ BP) ", Intl. J. Oncol. 8: 239-252; HT-144 (melanoma), DU-145 (prostate cancer), HeLa (cervix cancer), HL 60 (promyelocytic leukemia), MDA-MB-231 (breast cancer), SK-Br-3 (breast cancer), T47D (ductal breast cancer), A559 (Lung cancer) were obtained from American Type Culture Collection (Rockville, MD) and cultured in prescribed media. The effect of DIME was tested in cultures with a starting cell density of 2 x 10 ⁴ cells per cm ² and the comparison of the effect of DIME on cell growth (intact cells are identified by trypan blue exclusion) became 72 hours after direct cell counting after trypsinization in a hemocytometer Drug addition studied.

9.5 Tumorigenic effect from E-ras 20 cells

The tumorigenic effect of E-ras 20 cells in athymic nude mice was investigated as previously described. Bauer et al., 1996, "Modification of Growth Related Enzymatic Pathways and Apparent Loss of Tumorigenicity of a Transformed Bovine Endothelial Cell Line by Treatment with 5-iodo-6-amino-1,2-benzopyrone (INH ₂ BP)" , Intl. J. Oncol. 8: 239-252. The anti-tumorigenic effect of DIME in vivo was studied in athymic mice inoculated with 10 ⁶ MDA-MB-231 cells as in the tumorigenic activity assay. In about 10-14 days, when subcutaneous tumors appeared, DIME treatment consisting of po administration of a DIME suspension (once a day) was started and continued for 28-32 days, as indicated al., 1996, "Induction of tumor apoptosis by methyl-3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME)", Abstract No. 102, Int. J. Oncol. 9: Supplement 829.

9.6 Assays for Quantification the colony formation

Assays for quantifying colony formation were performed as indicated, Vidair et al., 1986 "Evaluation of a Role for Intracellular Na ⁺ , K ⁺ , Ca ²⁺ and Mg ²⁺ in Hyperthermic Cell Killing" Radiation Res. 105: 187-200.

9.7 DIME Sepharose affinity column

EAH-Sepharose (Pharmacia, Piscataway, NJ, USA) (2.0 g wet) was washed with water and the solvent against 60% DMF (aq) exchanged. The wet cake was redissolved in 60% DMF (1.0 ml). which contains the carboxylic acid derivative of DIME (Compound 6) (60 mg) and a tenfold excess of N, N'-dicyclohexylcarbodiimide (Sigma) and the suspension was at ambient temperature Carefully turned for 16 hours. The Sepharose beads were then collected on a sintered glass filter and successively with DMF, dioxane, DMF, aqueous DMF (66%, 50% and 33%) and finally washed with water. Based on the UV absorption spectrum of the substituted beads the content of DIME groups was in the range of 1-2 mmol per ml wet cake.

9.8 Metabolism of [ ¹⁴ C] -DIME

(a) Mouse tissue homogenate (brain, kidney, liver, lung) consisting of 0.2 g tissue in 1.0 ml homogenization buffer (50 mM Tris (pH 7.4), 400 mM NaCl, 10 mM MgCl ₂ , 1 mM EDTA , 0.5% NP-40, 0.5 mM PMSF) was mixed with 1.0 ml each of MES buffer (100 mM, pH 6.5), 20 μM [ ¹⁴ C] -DIME (10.55 mCi / mmol) and the mixtures were incubated for 4 hours at 37 ° C. Then ethyl acetate (1.5 ml), ammonium sulfate (900 mg) and 60% perchloric acid (160 μl) were added sequentially to each tube, vortexing after each addition. After standing for ten minutes, the phase separation was improved by centrifugation on the laboratory bench. The upper (ethyl acetate) phase was stripped off and the lower aqueous phase and the interfacial material were extracted with a second portion of ethyl acetate (1.5 ml). The ethyl acetate extracts (combined) contained more than 90% of the total cpm present in the original incubate (approximately 4 x 10 ⁵ cpm). The extracts were evaporated to dryness using an N ₂ stream, the respective residues were taken up in ethyl acetate (100 μl) and aliquots (10 μl) were applied to analytical silica gel TLC plates (flexible Whatman PE SIL G / UV plates, 250 μm thickness, 10 cm × 20 cm) spotted and with 3: 1: 0.8 Vol.:Vol.:Vol. n-hexane / ethyl acetate / ethanol developed. Analyte bands including [ ¹⁴ C] DIME as the reference standard were visualized by autoradiography. Additional reference standards (non-radioactive DIME and its carboxylic acid analogue) were visualized on the plates under UV light. (b) Metabolism of [ ¹⁴ C] -DIME in cells in culture: Cells (2-5 x 10 ⁶ in each experiment) were incubated with [ ¹⁴ C] DIME, then homogenized and assayed for metabolites as in (a).

9.9 Intracellular concentration from DIME

Monolayer cultures in 9.6 cm ² wells (3 ml of medium) were exposed to [ ¹⁴ C] -DIME (10.55 mCi / mmol) for 24 hours, then washed seven times with 1 ml of medium containing unlabelled DIME. The cells were dissolved in 1 ml of 4% Na ₂ CO ₃ solution and 0.2 M NaOH and the radioactivity measured by scintillation counting. Cell volume was determined by hematocrit and cell counting from parallel wells treated with unlabelled DIME.

9.10 Assay for DNA sections

Of the Assay for DNA sections as a sign of apoptosis was done via the Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Reaction, as specified by the manufacturer of the assay kit (Boehringer Mannheim, Indianapolis, IN, USA, cat. No. 168-4817).

9.11 Results

Of the Carboxylesterase inhibitor Bis [p-nitrophenyl] phosphate (BNPP), Heymann et al., 1968, "Inhibition of phenacetin and acetanilide-induced methemoglobinemia in the rat by the carboxyl esterase inhibitor of bis- [p-nitrophenyl] phosphate ", Biochem. Pharmacol. 18: 801-811, was obtained from Sigma.

The structural specificity of DIME and its homologs and analogs can be assessed by examining Table 7 and Table 8. We have prepared 17 new structural analogues of DIME and compared the anti-tumor efficacy of 10 of the compounds (Table 8), which seems sufficient to draw some general conclusions. The selected biological assay was the determination of the antitumor effect of the drugs on the tumorigenic effect of E-ras cells in nude mice in vivo, Bauer et al., 1996, "Modification of Growth Related Enzymatic Pathways and Apparent Loss of Tumorigenicity of a Ras-Transformed Bovine Endothelial Cell Line by Treatment with 5-iodo-6-amino-1,2-benzopyrone (INH ₂ BP) ", Intl. J. Oncol 8: 239-252. E-ras 20 cells (10 ⁵ or 10 ⁶ ) were incubated with 10 μM DIME or analogs for 4 days, then 10 ⁵ and 10 ⁶ cells were inoculated subcutaneously in nude mice (5 per test) and tumor formation rates quantified by direct tumor volume measurements (see Figure 2) itself is shown ( 4 ), the tumor formation was completely abolished. Identical antitumorigenic experiments were performed with nine DIME analogs, and, taking the effect of DIME as 100% (comparing tumor sizes on day 25), efficacy against tumors was calculated as percent of DIME potency. It should be noted that E-ras 20 cells are relatively less sensitive to DIME than human tumors; therefore, these results serve only as a basis for comparison of DIME analogs. The results summarized in Table 8 illustrate that substituents in R ₁ and R ₂ determine anti-tumor activity. This effect is particularly noticeable when _CH3 O, EtO, n-PrO, nBuO compares in _R1, suggesting that side chain parameters, not the binding of the "thyroxine-like" core structure itself, appear to confer pharmacological specificity.

This conclusion is corroborated by preliminary protein binding studies with the DIME-Sepharose affinity column. In this affinity column, DIME was covalently bound to a matrix on R ₂ ; therefore, this DIME derivative would have significantly less tumoricidal activity than DIME, analogous to the results in Table 8. (See Kun et al., U.S. Patent Application Serial No. 08 / 655,267, filed June 4, 1996, "Method of however, the percolation of cell extracts through this column resulted in protein binding to the affinity matrix, and one of the absorbed proteins was identified as tubulin determined by the Centricon method as previously indicated, Bauer et al., 1996, "Modification of Growth Related Enzymatic Pathways and Apparent Loss of Tumorigenicity of a ras-Transformed Bovine Endothelial Cell Line by Treatment with 5-iodo-6. amino-1, 2-benzopyrone (INH ₂ BP) ", Intl J. Oncol 8: 239-252 Calculated from Scatchard plots, k _D values of 10 ⁹ to 10 ^{10 were recorded} in cell extracts of proteins present, even with BSA, indicating a presumably hydrophobic association of DIME with various proteins. Based on the results with the DIME-Sepharose affinity column, we attribute this binding to the "core" structure of DIME.

TABLE 8 Inhibition of E-ras Tumorigenicity: Effectiveness of DIME and some structural analogues

The efficacy of DIME analogues on the tumorigenic effect of E ras 20 cells in athymic mice in vivo was compared to that of DIME (Compound 1), taken as 100, with the magnitude of tumor reduction determined on day 25. Pretreatment with DIME or its analogues (Table 7) was at 10 μM 4 days prior to subcutaneous inoculation. ^{a is} assigned as in Table 7.

The relative potencies of the DIME analogs can be deduced from Table 8, however, the present experiments focused primarily on the cell-destroying effect of DIME itself To define experimental systems suitable for more detailed analyzes of DIME analogues. If one considers the antitumotigenic effect of DIME ( 1 ) on in vivo conditions, it is shown that feeding DIME to nude mice produces 70-85% tumor regression in 25-35 days by daily doses of 0.25 to 1.0 g / kg per os, Kun et al., 1996, "Induction of tumor apoptosis by methyl-3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME)", abstract no. 102, Int. J. Oncol. 9: Supplement 829. These large doses produced no perceptible toxicity for the probable cause described below. Since 0.25 g / kg and 1.0 g / kg DIME had the same antitumor effect, a dose-response relationship was not visible. This paradox can be explained by selective drug uptake into tumor cells in vivo, as discussed.

In the present study, we focused on microscopic analysis of cell killing. As it is in 5 , the colony-forming ability of MDA-MB-231 cells was greatly reduced by DIME in a concentration range of 1-2 μM in 10 days. When DIME was removed prior to the end of the eight hour incubation with cells, cell killing was prevented and drug-induced morphological changes were completely abolished. Replating cells washed free of DIME for up to 8 hours after preincubation with 1-10 μM DIME resulted in a reuptake of normal cell growth and replication. The nature of crucial processes that produce an irreversible pathway leading to cell death after eight hours of drug treatment is yet unknown and is the subject of further investigation. However, one of the easily identified causes of the final cell death induced by DIME is drug-drug-dependent DNA breaks, as described in US Pat 6 is illustrated.

One striking feature of DIME is its apparent selectivity for tumors in vivo. We performed drug metabolism and cellular DIME uptake assays to further characterize this property. Incubation of cells in culture with [ ¹⁴ C] -DIME under the conditions previously described, Bauer et al., 1996, "Modification of Growth Related Enzymatic Pathways and Apparent Loss of Tumorigenicity of a ras-Transformed Bovine Endothelial Cell Line by Treatment with 5-iodo-6-amino-1,2-benzopyrone (INH ₂ BP) ", Intl. J. Oncol. 8: 239-252, resulted in the cellular uptake of the drug into a form that can not be washed out of cells with PBS containing non-radioactive DIME at the same concentration added extracellularly during incubation (see 2). The drug uptake rate is significantly higher in cells (eg, MDA-MP-231) that are most easily killed by DIME compared to fewer DIME-sensitive cells (Table 9). We developed a transformed normal renal cell phenotype, CV-1 cells, which show a loss of contact inhibition and a rapid doubling time (12 hours compared to 54 hours of the non-transformed phenotype). As shown in Table 9, the tumorigenic transformed cells generally absorbed DIME more rapidly than non-transformed cells (CV-1) or cells that were less susceptible to DIME killing (eg, E-ras 20). Established cell lines have gone through an immortality crisis, so they are not exactly physiologically working cells, and our results for drug uptake and the apparent lack of DIME toxicity in vivo suggest that normal cells in intact animals can not take up any or very little DIME. On the other hand, homogenates of normal mouse organs efficiently metabolize (de-esterify) DIME as illustrated in Table 10. The highest rate of "DIME esterase" activity occurred in brain homogenate.

TABLE 9 Recording of DIME

• ^a Verbindung 6 in Tabelle 7. ^b In nmol, Isoliert durch Dünnschichtchromatographie, ausgehend von 20,0 mol [¹⁴C]-DIME inkubiert in 2,0 ml Homogenisat bei 37°C für 4 Stunden. Die Cpm-Werte weisen eine Streuung von ±2% auf. Die spezifische Radioaktivität des [¹⁴C]-DIME war 20470 cpm/nmol.

Cellular concentration of DIME by four cell types after a 24-hour incubation (see procedure). ^a μM; ^b The concentration of DIME was chosen which causes 100% growth inhibition, leaving 20 to 50 x 10 ⁴ adherent cells / cm ² . TABLE 10 Esterase cleavage of DIME to its carboxylic acid ^a in various tissue homogenates

• ^a Compound 6 in Table 7. ^b In nmol, isolated by thin layer chromatography, starting from 20.0 mol of [ ¹⁴ C] -DIME incubated in 2.0 ml of homogenate at 37 ° C for 4 hours. The Cpm values have a scatter of ± 2%. The specific radioactivity of the [ ¹⁴ C] -DIME was 20470 cpm / nmol.

The correlation of drug metabolism and tumoricidal activity of DIME has also been demonstrated in human lung tumor cell experiments (A-549). As it is in 4 A-549 cells readily cleave the ester bond (R ₂ ) in DIME, while inhibiting esterase activity with bis [p-nitrophenyl] phosphate, Heymann et al., 1968, "Inhibition of Phenacetin and Acetanilide-Induced Methemoglobinemia in the Rat by the Carboxyl Esterase Inhibitor of Bis [p-Nitrophenyl] Phosphates, Biochem. Pharmacol. 18: 801-811, led to much more effective cell killing by DIME on A-549 cells, as described in US Pat V sees. A comparison of the cell growth inhibitory effect of DIME on various cancer cells expressed in DIME concentration causing 50% cell inhibition in 3 days is summarized in Table 11. A time course of the effect of DIME at 0.5, 1.0 and 2.0 μM final concentration on MDA-MB-231 cells is in 6 illustrated.

Table 11 Effect of DIME on various human cancer cell lines

For DIME challenge experiments, cells were seeded in 2 cm ² wells at a density of 2 × 10 ⁴ cells / cm ² . DIME at various concentrations was added to the medium at the time of sowing. The cell cultures were then incubated for 3 days (at 37 ° C in 5% CO ₂ atmosphere).

DIME is a unique tumoricidal molecule in that it does not appear to exhibit macroscopically observed toxicity in vivo except for tumor cells that grow in intact animals. From drug uptake assays, it seems obvious that cells susceptible to cell death by DIME are the most eager to take the drug. The replication of non-cancer cells growing in cultures is also inhibited to varying degrees by DIME (not shown) while the drug shows no toxicity in vivo. Therefore, it seems likely that the permeability of the cells for DIME in cell cultures of in vivo cells. The reasons for this apparent tumor specificity may well depend on differences between a hitherto unknown cell membrane property of cultured cells and cells growing in tissues of animals, an object of further investigation. This apparent permeability difference for DIME between (both tumor and non-tumor) cells grown in cell cultures and in vivo cells does not apply to tumor cells growing in vivo, Kun et al., 1996, Induction of tumor apoptosis by methyl-3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME) ", Abstract No. 102, Int. J. Oncol. 9: Supplement 829, as they are killed in vivo by DIME feeding, Kun et al., 1996, "Induction of tumor apoptosis by methyl-3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME)" , Summary No. 102, Int. J. Oncol. 9: Supplement 829. The DIME doses given in vivo (0.25-1.0 g / kg) greatly exceed the extracellular tumoricidal concentration obtained with cell cultures (0.5-2.0 μM), demonstrating that tumor cells can in vivo take up a small portion of the in vivo administered DIME, and tumor cells growing in vivo appear to have retained the same sensitivity for DIME as in cell cultures, ie tumor cells in culture and in vivo show similar drug uptake. The similarity of tumor cell membranes in vivo to cell lines growing in cell cultures appears to be a new propensity of tumor cells requiring further analysis. In addition to the apparent in vivo selectivity for DIME penetration into tumors, tumor cells differ from normal cells, which generally have no detectable DIME esterase activity except in the case of A-549 cells (lung cancer) (not shown).

Investigations on these cells ( 4 . 5 ) strikingly illustrate that DIME itself, not its esterase-generated metabolite, is the tumoricidal molecule. As can be seen from Table 8, replacement of the methyl ester group of DIME (R ₂ ) with the longer chain ethyl ester group or with amide groups still significantly enhances the antitumor activity of certain DIME analogs on E-ras 20 cells. In this study, only DIME has been analyzed in some detail, but various "effective" DIME analogs also deserve further attention, as it is possible that they may have cellular and in vivo effects that can be exploited for an additional chemotherapeutic use , As far as the iodine atoms in DIME and its analogues are concerned, these bulky atoms undoubtedly impose conformational restrictions between the two benzene rings and, at the same time, crucial determinants of cell penetration may be present.

EXAMPLE 10: Effect of DIME on cell and nuclear morphology

The The following provides further guidance regarding the broader concept the use of thyroxine analogues when treating a wide range of cancerous ulcers ready. We have reported that DIME is a strong trigger of the Cell death in tumor cells in cell cultures and in vivo. The selective Tumoricidal effect will most likely by selectively penetrating this drug into tumor cells in vivo explains, Mendeleyev et al., 1997, "Structural Specificity and Tumoricidal Action of Methyl 3,5-Diiodo-4- (4'-Methoxyphenoxy) Benzoate (DIME), "Intl. Oncol., In press. As described here, the effect of 1 to 4 DIME on tumor cells to cytological changes and mitotic blockade, what the involvement of different biochemical active sites of DIME predicts. It was important before analyzing the bodies on one biochemical level first through the cellular mode of action of DIME to define cytometric procedures. The present example is concerned with the effect of DIME on cell and nuclear morphology and the Progression through mitosis.

10.1 Microscopic Morphology

The microscopic morphology of E-ras cells was investigated by previously reported techniques, Mendeleyev et al., 1997, "Structural Specificity and Tumoricidal Action of Methyl 3,5-Diiodo-4- (4'-Methoxyphenoxy) Benzoate (DIME),""Intl. J. Oncol., In press; Bauer et al., 1996, "Modification of Growth Related Enzymatic Pathways and Apparent Loss of Tumorigenicity of a Transformed Bovine Endothelial Cell Line by Treatment with 5-iodo-6-amino-1,2-benzopyrone (INH ₂ BP)" ,

10.2 Preparation for Visualization of the M phase

The MDA-MB-231 cell line was obtained from ATCC (Rockville, MD, USA) and the cells were grown in T-75 flasks at 37 ° C in Minimum Essential Medium-α supplemented with 10% FCS. For controls that did not receive DIME, 0.1 μg / ml colcemid was added to the cultures 4 hours prior to collection of the metaphase-blocked cells and preparation of cell spreads. The effects of DIME and colcemid are indistinguishable in terms of cell cycle blockade in the M phase; therefore, when the effects of DIME on metaphase spreads were tested, no colcemid was added. Cells (10 ⁷ ) were removed from 5 min ger T-75 Trypsinisierung with 0.025% trypsin isolated and sedimented by centrifugation, resuspended and kept 10 min. In 10 ml 75 mM KCl solution at 37 ° C, sedimented again and in 10 ml of methanol-acetic acid with four successive changes of methanol Acidified. A subset of the final cell suspension (100 μl) was dropped on ethanol-cleaned slides and air dried.

10.3 In situ hybridization

Samples specific for human chromosomes were generated by ONCOR (Gaithersburg, MD, USA). Hybridization was performed by a modification of the method described by Pinkel et al., 1986, "Cytogenetic Analysis Using Quantitative High-Sensitivity Fluorescence Hybridization," Proc. Natl. Acad. Sci. USA 83: 2934-2938. The cells mounted on the slide were treated with pepsin (20 μg / ml in 0.01 N HCl) at 37 ° C for 10 minutes and then dehydrated in succession with 70%, 85%, and 100% ethanol, then DNA by immersion in 70% formamide followed by 2X standard salt citrate (1X SSC is 0.5M NaCl, 0.015M sodium citrate, pH 7.0) for 2 min. At 70 ° C, and denatured Ethanol dehydrated as described above. The hybridization mixture in a 10 μl total volume consisted of 50% formamide, 2 × SSC, 10% dextran sulfate, 0.5 μg herring sperm DNA and 1-5 μg proteinase K-treated human placental DNA. Both herring sperm DNA and human placental DNA were previously sonicated to 200-600 base pair fragments and 40 ng of digoxigenylated sample DNA (denatured at 70 ° C for 5 min.) And incubated for 1 h at 37 ° C. This mixture was placed on slides containing the fixed cells, sealed under cover slip, and incubated at 37 ° C for 2-3 days. After hybridization was complete, the slides were replaced with three changes (3 x 5 min.) Of 50% formamide, 2 x SSC, pH 7.0, and two times in PN buffer (consisting of a mixture of 0.1 M NaH ₂ PO ₄ and 0.1 M NaHPO ₄ , 0.1% Nonidet P-40, pH 8.0) at 45 ° C, then with 5 μg / ml antidigoxigen in FITC, 2 μg / ml rabbit anti-sheep FITC ( Boehringer Mannheim) in PNM buffer (which contains 5% nonfat dry milk containing 0.02% sodium azide after centrifugation to remove solids) for 20 min at room temperature, after each incubation, for 3 min. In PN. Buffer and stained for DNA with 0.4 μM DAPI (4,6-diamino-2-phenylindole) in Antifade solution (Vector labs, Burlingame, CA, USA). The slides were viewed in a Zeiss fluorescence microscope equipped with a multiple band pass filter (Chroma Technology, Brattleboro, VT, USA) to determine the number of FISH signals in each nucleus.

10.4 Time-lapse video microscopy

cell in sealed T-25 tissue culture flasks were placed in a temperature-controlled Incubation chamber, which is built so that it is an inverted Phase contrast microscope includes. The chamber was surrounded by Room light shielded. Every 5 min, the microscope light was turned on for 12 sec. turned on while what time a picture is taken by an imaging system made by Compix, Inc. (Mars, PA, USA) was detained. The picture sequences were analyzed with software from the same company.

10.5 Flow cytometry

Cells exposed to various treatments were grown to confluency, trypsinized and washed with phosphate buffered saline (PBS). For core analysis, the cells were fixed in Vindelov citrate cell buffer: sucrose 250 mM, trisodium citrate · 2H ₂ O 40 mM, DMSO 50 ml in 1000 ml solution, pH 7.6. Normal human lymphocytes obtained from blood were used as controls. Each cell sample and control was counted with a hemocytometer and the cell concentration was adjusted to 2 x 10 ⁶ cells / ml. Two million cells from each cell line in a total volume of 2 ml were washed twice with fresh PBS, treated with 200 μg / ml RNAase at 37 ° C for 30 min and stained with 10 μg / ml propidium iodide for 45 min. Flow cytometric analysis was performed on a FACScan benchtop flow cytometer (Becton-Dickinson, San Jose, CA) equipped with an air-cooled argon laser tuned to 488 nm. Twenty thousand events were collected six parameter list mode value data for analysis and archiving. Two scattered light parameters (forward and to the side), propidium iodide fluorescence measured at 575/26 nm and above 620 nm, and doublet resolution by fluorescence pulse width and area were detected at 1024 data channel resolution. The detection threshold has been set to propidium iodide positive events above channel 100. Hiding to eliminate small foreign objects, large clumps and cell doublets was done after capture.

10.6 Immunocytochemistry

cell were fixed in methanol for 5 min. at -20 ° C. The primary antibody was monoclonal anti-β-tubulin the mouse (Amersham) and the goat secondary antibody to fluorescein isothiocyanate from Cappel (Durham, NC, USA). The DNA the cores became in ten minutes Incubation in 0.05 μg / ml 4 ', 6-diamidino-2-phenylindole (DAPI stained. The stained Cells were probed with either a Zeiss 40x Plan neofluar or a Nikon 60 × PlanApo lens considered. To classify the cells in the mitosis phase were Prophase and metaphase in a single category, since it was difficult, the late To distinguish prophase from the metaphase.

10.7 results

The effects on cell morphology after 18-24 hours incubation of E-ras 20 cells with 4 μM DIME are reported in 10 illustrated. The tumor cells expanded and numerous micronuclei appeared, as can be seen especially at 300x magnification. The nature of these cytological changes was further investigated. Flow cytometric analysis ( 11 ) performed on human MDA-MB 231 breast cancer cell nuclei showed that cores having a G2 content of DNA had accumulated through 18-hour drug exposure, indicating M-phase blockage.

The above observation prompted us to investigate the kinetics of entry into mitosis in cells exposed to DIME. The cells were incubated in medium containing 1 μM DIME and monitored by time-lapse video microscopy as described in Materials and Methods. 12 Figure 12 shows images illustrating a 13-hour delay in mitosis followed by an irregular division into 6 daughter cells. In contrast, the control cells delayed about 0.5 hr in mitosis (see 13 ) and always shared, giving exactly 2 daughter cells (data not shown).

• ^aMitosen wurden während kontinuierlicher Einwirkung von DIME auf die Zellen durch Zeitraffervideomikroskopie überwacht; ^bVerschmelzung der Tochterzellen trat normalerweise innerhalb Minuten nach der Zytokinese auf und beteiligte 2–5 Tochterzellen.

Time-lapse video microscopy also allowed us to monitor the fate of the daughter cells resulting from the irregular cell division described above. Twenty percent of mitosis seen in the presence of 1 μM DIME yielded daughter cells fused (Table 12), often large polynuclear cells of the type to be found in 10 sees, were received. Control cells showed no such division, followed by fusion. TABLE 12 DIME triggers the fusion between daughter cells after mitosis

• ^a Mitoses were monitored by continuous exposure of DIME to the cells by time-lapse video microscopy; ^b Daughter cell fusion usually occurred within minutes of cytokinesis and involved 2-5 daughter cells.

The cell division, which took place in the presence of 1 μM DIME, was examined to quantitatively estimate the number of daughter cells resulting from each mitosis ( 14 ). The number of daughter cells ranged from 1 to 6 per mitosis event. In contrast, the control cells always shared (33/33), giving 2 daughters. Thus, the drug-induced long delay in mitosis described above was followed by a highly abnormal cell division pattern.

15 Figure 11 shows the rates at which the cells incubated in 1 μM DIME and the control cells entered into mitosis. The curves are nearly coincident, indicating that DIME did not affect the speed at which the cells went through the interphase. In contrast, the average time that each cell spent in mitosis increased more than 20-fold by the drug gert ( 13 ). The fact that the cells blocked by DIME were indeed in mitosis in a circular configuration was due to their truncated and expanded chromatin ( 16 ) and abnormal Mitosespindeln ( 17 ) approved.

Chromosome analyzes by in situ hybridization of the metaphase nuclei were performed with investigations specific for chromosomes 1, 2, 7, 11 and 19. They all gave similar results; therefore only chromosome 19 is illustrated. Representative results are for chromosome 19 in the 16 A, B and C shown. In all cases, MDA-MB-231 (human breast cancer) cells were used. 16A shows in situ hybridization on chromosome 19. The 18-hour exposure to 1 μM DIME had no detectable effect; that is why the 16A and 16B both control and drug treated cells. 16B is DNA staining (DAPI), demonstrating the agreement of DNA staining with chromatin while 16A is stained for chromosome 19. However, the 5-day drug exposure to the cells (1 μM DIME) induces a large accumulation of metaphase chromatin in some cells with approximately 40 chromosome 19 signals and over 100 chromosomes ( 16C ). No evidence of chromosome breakage could be obtained, even though this drug treatment ultimately kills cells, Mendeleyev et al., 1997, "Structural Specificity and Tumoricidal Action of Methyl 3,5-Diiodo-4- (4'-Methoxyphenoxy) Benzoate (" DIME), "Intl. J. Oncol. 10: 689-695.

The structure of the mitotic spindle after 18 hours exposure to 1 μM DIME is in 17 shown. 17A is the tubulin-stained mitotic spindle in non-drug-treated MDA-MB-231 cancer cells. 17B shows the changes after 18 hours of drug exposure, which consisted of conspicuous abnormalities of tubulin distribution. These multi-centered structures were observed after 5 days of 1 μM DIME ( 17C ) expanded even more. The cellular multicentric distribution of tubulin ( 17E and 17C ) appears to be similar to the nuclear formation of tubulin by added centrosomes in vitro, as shown by Mitchison et al., 1984, Microtubule Assembly Nucleated by Isolated Centrosomes, Nature 312: 232-237. However, we were unable to detect an abnormal number of centrosomes by immunofluorescence (data not shown); thus, the visible multicentric core formation of tubulin after 18 hours of DIME treatment (1 μM) can not be directly related to the centrosomes. These images do not predict whether 1 μM DIME induces tubulin polymerization or inhibits re-polymerization or not.

In preliminary investigations (data are not shown) we have also the behavior of certain spindle-associated proteins the immunocytological technique. Cdc-2 kinase was in Absence of 1 μM DIME not associated with the spindle, but the 18-hour impact of the drug on the cells showed induction of cdc-2 kinase spindle binding. The Drug treatment changed not the pattern of staining by antiserum for the centrosomal protein pericentrin, since only two foci were seen. The cyclin B microtubule spindle association also remained unchanged. however the drug induced an abnormal organization of the spindle pole centers, which are still bound to cyclin B. The protein phosphatase 2a (pp2a) spindle association was just like that of pericentrin or cyclin B not through the drug treatment (1 μM for 18 hrs.) affected.

These exploratory investigations include the basis of other cell biological Research. In preparatory trials also became the possible role of phosphatases in protein spindle associations checked. The 18-hour Influence of 100 nM extracellular added okadaic acid lifted only the cdc-2 kinase and pp2a spindle association on, indicating the Involvement of protein phosphatases suggests (data are not shown).

These Results seem to be between early Effects of 1 μM DIME, which is within 18-24 hours take place after drug exposure, and late episodes to distinguish, which one sees here in the Chromosomenzahlen, the develop after 5 days of drug treatment. However, that is it probably that late Events reflect only a cascade of cellular responses, the binding of DIME to certain cellular sites be initiated. morning Close events noticeable cytological changes in particular the appearance of large multinucleated cells. It is well known that the formation of micronuclei after radiation damage that results from the failure of acentric chromosomal fragments is due to a nuclear migration to the poles during anaphase the absence of kinetochores and microtubule spindle attachment J. Bedford, 1991, "Sublethal Damage, Potentially Lethal Damage, and Chromosomal Aberration in Mammalian Cells exposed to Ionizing Radiation, "J. Radiation Oncol. Biol. Phys. 21: 1457-1469. time-lapse studies have shown that vinblastine sulfate also metaphase-blocked polynuclear Cells, Kirshan, 1968, "Time Lapse and Ultrastructure Studies on the Reversal of Mitotic Arrest Induction by Vinblastine Sulfate in Earl's L Cells, "J. Natl.

Cancer Institute 41: 581-595 following the results presented here ( 10 ), but vinca alkaloids also show serious toxic effects in animals, whereas DIME does not. Overexpression of protein phosphatase 2a (pp2a) also induces multiple nucleus formation, Wera et al., 1995, "Deregulation of Transitional Control of the 65 kDa Regulatory Subunit (PR65 Alpha) of Protein Phosphatase 2A Leads to Multinucleated Cells," J. Biol. Chem 270: 21374-21381, which clearly demonstrates that the mechanism of development of this complex process (multiple nucleation) may result in a variety of likely contiguous enzymatic activities. These results show activation of pp2a in DIME cells (lcl), an effect likely due to a direct effect of DIME on this enzyme. The involvement of various cellular enzymes in the mode of action of DIME is the subject of a separate biochemical report.

One of the most easily observed cellular phenomena induced by DIME is blockade in the M phase ( 11 and 13 ), the effect of colcemid or especially vinca alkaloids, which can be replaced by DIME, very similar. Late effects of DIME (after 5 days), as illustrated by DNA fluorescence hybridization with samples on chromosomes 1, 2, 7, 11 and 19, are the result of accumulation of chromosomes due to lack of cell division. DIME has no visible direct effect on DNA. The sections in double-stranded DNA ( 12 in Mendeleyev et al., 1997, "Structural Specificity and Tumoricidal Action of Methyl 3,5-Diiodo-4- (4'-Methoxyphenoxy) Benzoate (DIME)," Int. J. Oncology 10: 689-695, are downstream consequences of the interaction of DIME with cancer cells and most likely reflect the upregulation of DNA endonucleases which in DIME-treated cells by way of indirect activation of poly (ADP-ribose) glycohydrolase ( Footnote I in ref. 1) de-poly-ADP-ribosylated. Endonuclease activation may not be the only single cause of programmed cell death by DIME, as it is known that various molecular mechanisms can lead to apoptosis, Wertz et al., 1996, "Diverse Molecular Provocation of Programmed Cell Death," TIBS 21: 359-364. The development of an abnormal mitotic spindle in DIME-treated cells indicates an initiating cellular effect of DIME on the tubulin system, which is well known to play a crucial role in cytokinesis, Murray et al., 1993, "The Cell Cycle : An Introduction, Oxford University Press, New York.

There DIME a "hormonal inactive "thyroid hormone analogue is, the fascinating question comes up, whether metabolic precursors (or catabolites) of thyroid hormones in physiological differentiation maintenance, serving as "anti-malignant" correction regulators, play a role or not. A search for thyroid hormone metabolites with tumorizider Effect seems justified.

EXAMPLE 11: Effect on tubulin

In the following experiments, the hormone-inactive thyroid hormone analog, DIME, inhibits the GTP-dependent polymerization of MTP in 1 to 5 μM concentrations, as determined by an optical assay. This inhibition is critically dependent on the GTP concentration. The quantitative correlation between the concentrations of DIME and GTP under the conditions of a linear MTP polymerization rate follows a Michaelis-Menten kinetics and the inhibition is a "mixed" type, with km being changed simultaneously for GTP and U _max . Chemical analogues of DIME inhibit MTP polymerization in parallel with their antitumorigenic activity in vivo. The MTP site is one of the early cellular responses of DIME.

Exposure of 1 μM DIME to human breast cancer cells (MDA-MB-231) induced abnormal spindle structures within 18 hours of drug treatment; thus, putative DIME microtubule protein (MTP) interaction appears to be a component of early cellular drug responses, Zhen et al., 1997, "Cellular Analysis of the Mode of Action of Methyl-3,5-Diiodo-4 - (4'-methoxyphenoxy) benzoates (DIME) on tumor cells, Intl. J. Oncol. Abnormal spindle structures could be the result of a DIME-MTP interaction or reactions of DIME with constituents of the microtubule organizing center or with previously undefined systems sequentially or Since the time-dependent in-situ quantitative analysis of the MTP system is inadequate for initial rate measurement, we fitted the in vitro neurotubular assembly system as a model for a quantitative analysis of the interaction of DIME with MTP as described by Gaskin et al. , 1974, "Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules", J. Mol. Biol. 89: 737-758, and Kirschner et al., 1974, "Microtubules from mammalian brain: some properties of their depolymerization products and a proposed mechanism of assembly and disassembly ", Proc. Natl. Acad. Sci. USA 71: 1159-1163, this system is suitable for kinetic examination of the MTP assembly in vitro. The timing of the MTP assembly consists of initiation, propagation and termination steps, Gaskin et al., 1974, "Turbidimetric studies of the Biol 89: 737-758 The rate of propagation under defined conditions is sufficiently linear to allow kinetic analysis evaluated for DIME and GTP concentrations As we show here, inhibition of MTP assembly by DIME occurs in the same range of drug concentrations as is required to inhibit tumorigenesis in vivo or to inhibit cell replication or ultimately to induce cell death; Mendeleyev et al., 1997, "Structural specificity and tumoricidal action of methyl-3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME)" Int. J. Oncol. 10: 689-695 and Table 8 above; For example, the DIME-MTP interaction is most likely part of the apparent pleiotropic cellular mechanism of action of DIME.

11.1 Isolation of microtubule proteins (MTP)

The preparation of MTP and an optical assay for polymerization have been adopted from published procedures, Gaskin et al., 1974, "Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules", J. Mol. Biol. 89: 737-758; Tiwari et al., 1993, "A pH and temperature-dependent cycling method that doubles the yield of microtubule protein", Anal. Biochem. 215: 96-103. Bovine or rabbit brain was homogenized in an equal volume of ice-cold buffer containing 100 mM Pipes / K ⁺ (pH 7.4), 4 mM EGTA, 1 mM MgCl ₂ , 0.5 mM DTT and 0.1 mM PMSF and centrifuged at 39000 g for 1 hour at 4 ° C. To the supernatant, DMSO (8% final concentration) and GTP (1 mM final concentration) were added, followed by incubation at 37 ° C for 30 minutes. The microtubules were pelleted at 100,000 g at 37 ° C for 30 minutes. The pellets were incubated on ice for 15 minutes followed by resuspension in ice-cold PEM buffer (100 mM Pipes / K ⁺ (pH 6.9), 1 mM EGTA, 1 mM MgCl ₂ ). This cycle of warm polymerization and cold depolymerization was repeated once more and the cold, resuspended monomeric MTP (8-10 mg / ml protein) was used for the polymerization kinetics optical assay. Both rabbit and cattle brain provided identical MTP preparations.

The composition of the optical test is described in the legends of the figures and Table 12. The polymerization reaction was started by the addition of 100 μl of MTP solution (equivalent to 0.8-1.0 mg of protein) and the initial linear rates of increase in absorbance at 350 nm were monitored and incubated at 37 ° C (see 1 ) were recorded in a Perkin-Elmer 552 dual-beam spectrophotometer equipped with a thermostatically controlled cuvette holder.

11.2 Results and discussion

The accuracy of the optical investigation for the polymerization of MTP is in 18 illustrated. The experimental conditions were the same as those given in the legend of Table 7 except that the GTP concentration was 1 mM (right curve) and DIME was 4 μM (left curve). It is apparent that the rate of MTP polymerization proceeds in a linear fashion; thus, the conditions for maximum polymerization rates as defined by B. Berne, 1974, "Interpretation of light scattering from long rods", J. Mol. Biol. 89: 755-758, appear to be satisfied. Therefore, it is possible to determine the quantitative correlation between [DIME] and [GTP] by comparing the linear rate in the presence of different drug and activator concentrations. At 1 mM GTP concentration, increasing DIME concentrations increasingly inhibit MTP polymerization ( 18 ).

As it is in 20 1 μM DIME was quantitatively correlated with [GTP] and a double reciprocal plot produced a "mixed" type inhibition, Dixon et al., 1964 Enzymes, pp. 234-237, Acad. Press, Inc., New York. Both V _max and km values were changed by almost 50% k _m GTP from 6.7 μM to 14 μM while V _max decreased by nearly 50%. The simplest interpretation of a mixed inhibition based on the Briggs-Haldane equation (see 7) is that k ₂ , that is, the dissociation [ES] to E and P (product), is directly affected, which then both k _m and V _max changed. The exact nature of k ₂ is currently unknown and its determination requires the analysis of the reaction products of GTP hydrolysis, a work to be reported elsewhere. Allosteric modifications can also achieve similar results, Dixon et al., 1964 Enzymes, pp. 234-237, Acad. Press, Inc., New York. The purpose of the present experiments is to compare the efficacy of DIME with some of its analogues on MTP polymerization (see Table 12) and to correlate the results with cytopathological processes (eg inhibition of tumorigenesis in vivo).

There is an obvious correlation between the chemical structure of DIME and 7 of its analogs, which can act on MTP polymerization (Table 13), and its inhibitory activity on tumorigenicity in vivo (compare with Table 8 or Mendeleyev et al., Supra). , For example, the Substitution in R ₁ of CH ₃ O after EtO and n-BuO increasingly inhibits MTP polymerization, almost exactly in parallel with the decreasing anti-tumorigenic effect (compare with Table 8 or Mendeleyev et al., Supra). On the other hand, substitution in R ₂ from the methyl ester to the carboxylic acid completely abolishes the inhibitory effect on MTP polymerization, but halves only the antitumorigenic activity studied with E-ras 20 cells (see Table 8 and Figs 5 or Mendeleyev et al. above). It is possible that such quantitative differences may reflect cell type-specific changes.

TABLE 13 Effect of DIME and some analogues on microtubule assembly in vitro

The assay system consisted of 240 μl PEM buffer containing 8% DMSO and GTP (final concentration 1 mM), 10 μM final drug concentration (added in 3 μl), or solvent control and 0.6 mg MTP (60 μl). The microtubule assembly at 37 ° C was monitored at λ = 350 nm and initial rates were reported as mA ₃₅₀ / min. calculated. The control had an initial speed of 180 mA ₃₅₀ / min. on. The values are averages of duplicate examinations.

The Inhibition of MTP polymerization can be highly complex cellular consequences to have. In cytokinesis, this inhibition can reduce the tensile forces of Affect tubulins and the formation of a dividing groove necessary for cell division Burton et al., 1997, "Traction forces of cytokinesis measured with optically modified elastic substrates, Nature 385: 450-454, Inhibition of MTP Polymerization By DIME should be with the biochemical sites of this drug be correlated. Like opposite Mendeleyev et al .; above, DIME directly activates pp2-ase; therefore It is necessary to have this effect related to mitosis phenomena, which are induced by DIME to coordinate. For example, was recently, Kawabe et al., 1997, "HOXII interacts with protein phosphatase pp2a and pp1 and disrupts G2 / M cell cycle check point ", Nature 385: 454-458, that pp2-ase can regulate the G2 / M transition and pp2-ase also is a potential oncogene whose inhibition promotes oncogenesis. It is possible, activation of Pp2ase by DIME counteracts oncogenesis.

On Based on these experiments, it can be seen that thyroxine-type analogs, like DIME, which can block mitosis in cancer cells. The present invention makes a quick screening on such connections Use of these techniques and use of cell sorters, chromosome blot or another DNA analysis in cells.

Claims (8)

Use of a thyroxine analog, optionally in combination with a physiologically acceptable carrier, for the manufacture of a medicament for the treatment of a malignant tumor in a mammal, wherein the thyroxine analogue is characterized as being a compound capable of causing a 35% or greater inhibition of the initial rate of microtubule-protein assembly in vitro and wherein the thyroxine analogue has the formula

and pharmaceutically acceptable salts thereof, wherein: X = O, S, CH ₂ , carboxy or absent; Y = O or S; R ₁ = methyl or ethyl; R ₂ , R ₃ , R ₄ and R _{5 are} independently selected from H, (C ₁ -C ₄ ) -alkyl, (C ₂ -C ₄ ) -alkenyl, (C ₂ -C ₄ ) -alkynyl, hydroxyl, ( C ₁ -C ₄ ) alkoxy and halogen; and R ₆ , R ₇ , R ₈ and R _{9 are} independently selected from H, (C ₁ -C ₄ ) -alkyl, (C ₂ -C ₄ ) -alkenyl, (C ₂ -C ₄ ) -alkynyl, hydroxyl, (C ₁ -C ₄ ) alkoxy, halogen, NO ₂ and NH ₂ . Use according to claim 1, wherein the thyroxine analogue has the formula

and pharmaceutically acceptable salts thereof, wherein: X = O, S, CH ₂ , carboxy or absent; Y = O or S; R ₁ = methyl or ethyl; R ₂ , R ₃ , R ₄ and R _{5 are} independently selected from H, (C ₁ -C ₄ ) -alkyl, (C ₂ -C ₄ ) -alkenyl, (C ₂ -C ₄ ) -alkynyl, hydroxyl, ( C ₁ -C ₄ ) alkoxy and halogen; and R ₇ and R _{8 are} independently selected from H, (C ₁ -C ₄ ) alkyl, (C ₂ -C ₄ ) alkenyl, (C ₂ -C ₄ ) alkynyl, hydroxyl, (C ₁ -C ₄ ) Alkoxy, halogen, NO ₂ and NH ₂ . Use according to claim 1 or 2 wherein the thyroxine analog is methyl 3,5-diiodo-4- (4'-methoxyphenoxy) benzoate is. Use according to one the claims 1 to 3, wherein the thyroxine analogue is present in an amount which is effective, a regression to cause the malignant tumor. Use according to one the claims 1 to 4, wherein the malignant tumor is a carcinoma or a sarcoma. Use according to one the claims 1 to 5, wherein the drug is for oral administration is. Use of a thyroxine analog, optionally in combination with a physiologically acceptable carrier, for the manufacture of a medicament for the treatment of cancer, wherein the thyroxine analogue is the structural formula

and pharmaceutically acceptable salts thereof, wherein: X = O, S, CH ₂ , carboxy or absent; Y = O or S; R ₁ = methyl or ethyl; R ₂ , R ₃ , R ₄ and R _{5 are} independently selected from H, (C ₁ -C ₄ ) -alkyl, (C ₂ -C ₄ ) -alkenyl, (C ₂ -C ₄ ) -alkynyl, hydroxyl, ( C ₁ -C ₄ ) alkoxy and halogen; and R ₆ , R ₇ , R ₈ and R _{9 are} independently selected from H, (C ₁ -C ₄ ) -alkyl, (C ₂ -C ₄ ) -alkenyl, (C ₂ -C ₄ ) -alkynyl, hydroxyl, (C ₁ -C ₄ ) alkoxy, halogen, NO ₂ and NH ₂ . Use according to claim 7, wherein the cancer is a carcinoma or sarcoma.